Abstract
Use of the effects of plant exosomes on the immune system as immune system enhancers, silencers and modulators against diseases is disclosed. The plant derived exosomes are obtained from at least one portion of the plant selected from the group consisting of the entire plant, fruit, leaf, seed, root, or differentiated tissues like the plant's tissue culture medium, stem cell, waste material, shell or phloem. In the scope of the invention, the plant exosomes having immunomodulatory effects are used mainly in autoimmune diseases, and in cell, tissue, organ transplantations and in Graft Versus Host disease as immune system enhancers, suppressors or, if necessary, as modulators performing both of the first two functions.
Claims
1. Plant derived exosomes, wherein the plant derived exosomes are configured for a prevention and a treatment of diseases by activating, suppressing and modulating cells of an immune system.
2. The plant derived exosomes according to claim 1, wherein the plant derived exosomes are obtained from at least one portion of a plant selected from the group consisting of an entire plant, a fruit, a leaf, a seed, a root, differentiated tissues like the plant's tissue culture medium, a stem cell, a waste material, a shell and phloem.
3. The plant derived exosomes according to claim 2, wherein the plant derived exosomes are obtained from a plant tissue culture in order to produce exosomes at a concentration up to 5 times higher than the plant derived exosomes obtained from similar plants and to maintain a content and properties of produced exosomes for a predetermined time, thereby preventing the plant derived exosomes from being affected by effects of the farm, harvesting, and transport.
4. The plant derived exosomes according to claim 3, wherein cells in the plant tissue culture are transfected to produce proteins, and the proteins enhance, suppress or modulate the immune system.
5. The plant derived exosomes according to claim 1, wherein the plant derived exosomes are isolated by an isolation method selected from the group consisting of an isolation by two phase liquid system, a graduated centrifuge, an ultrafiltration, chromatographic methods, a polymer based isolation and an isolation by microbeads.
6. The plant derived exosomes according to claim 1, wherein the plant derived exosomes are used in autoimmune diseases, and in cell, tissue, organ transplantations, in Graft Versus Host disease, and diseases affecting the immune system as immune system enhancers, immune system suppressors or immune system modulators, the diseases affecting the immune system comprise rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus, sclerosis, Sjogren's syndrome, Type 1 Diabetes, allergic asthma, Wegener granulomatosis, Multiple Sclerosis, Crohn's disease, psoriasis, Graves' disease, Celiac Disease, alopecia areata (pelade), central nervous system vasculitis, Hashimoto's thyroiditis, myasthenia gravis, Goodpasture's syndrome, autoimmune hemolytic anemia, Guillan-Barre syndrome, polyarteritis nodosa, idiopathic thrombocytic purpura, temporal arteritis, primary biliary cirrhosis, Addison's Disease, ankylosing spondylitis, Reiter's syndrome, Takayazu's arteritis, and vitiligo, wherein the immune system modulators perform functions of the immune system enhancers and the immune system suppressors.
7. The plant derived exosomes according to claim 1, wherein the plant-derived exosomes are administered orally, intranasally, intravenously, intramuscularly, intradermally, topically, intraperitoneally, and by an injection for administering an effective dose of selected plant exosomes to a patient to treat immune system-mediated diseases.
8. The plant derived exosomes according to claim 1, wherein the plant derived exosomes are configured for carrying immunomodulatory drugs.
9. The plant derived exosomes according to claim 1, wherein the plant derived exosomes are configured as adjuvants in vaccination applications.
10. The plant derived exosomes according to claim 1, wherein the plant derived exosomes are configured as a nutritional supplement for a purpose of modulating the immune system.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0051] “Use of Plant Exosomes for Showing Modulatory Effects on Immune System Cells” developed to fulfill the objective of the present invention is illustrated in the accompanying figures wherein,
[0052] FIG. 1A shows the graphical representation of the characterization of the exosome isolated from the Warty-Leaved Rhubarb plant in the scope of the present invention (flow cytometry graph measured by a control group, by flow cytometry device); FIG. 1B shows the graphical representation of the characterization of the exosome isolated from the Warty-Leaved Rhubarb plant in the scope of the present invention (flow cytometry graph measured by CD9 antibody, which is an exosome characterization marker, by flow cytometry device); FIG. 1C shows the graphical representation of the characterization of the exosome isolated from the Warty-Leaved Rhubarb plant in the scope of the present invention (flow cytometry graph measured by HSP70 antibody, which is an exosome characterization marker, by flow cytometry device); FIG. 1D shows the graphical representation of the characterization of the exosome isolated from the Warty-Leaved Rhubarb plant in the scope of the present invention (flow cytometry graph measured by CD63 antibody, which is an exosome characterization marker, by flow cytometry device); FIG. 1E shows the graphical representation of the characterization of the exosome isolated from the Warty-Leaved Rhubarb plant in the scope of the present invention (exosome diameter measurement graph);
[0053] FIG. 2A shows the graphical representation of the characterization of the exosome isolated from Celery in the scope of the present invention (flow cytometry graph measured by a control group, by flow cytometry device); FIG. 2B shows the graphical representation of the characterization of the exosome isolated from Celery in the scope of the present invention (flow cytometry graph measured by CD9 antibody, which is an exosome characterization marker, by flow cytometry device); FIG. 2C shows the graphical representation of the characterization of the exosome isolated from Celery in the scope of the present invention (flow cytometry graph measured by HSP70 antibody, which is an exosome characterization marker, by low cytometry device); FIG. 2D shows the graphical representation of the characterization of the exosome isolated from Celery in the scope of the present invention (flow cytometry graph measured by CD63 antibody, which is an exosome characterization marker, by flow cytometry device); FIG. 2E shows the graphical representation of the characterization of the exosome isolated from Celery in the scope of the present invention (exosome diameter measurement graph);
[0054] FIG. 3A shows the graphical representation of the characterization of the exosome isolated from Pomegranate in the scope of the present invention (flow cytometry graph measured by a control group, by flow cytometry device);
[0055] FIG. 3B shows the graphical representation of the characterization of the exosome isolated from Pomegranate in the scope of the present invention (flow cytometry graph measured by CD9 antibody, which is an exosome characterization marker, by flow cytometry device); FIG. 3C shows the graphical representation of the characterization of the exosome isolated from Pomegranate in the scope of the present invention (flow cytometry graph measured by HSP70 antibody, which is an exosome characterization marker, by flow cytometry device); FIG. 3D shows the graphical representation of the characterization of the exosome isolated from Pomegranate in the scope of the present invention (flow cytometry graph measured by CD63 antibody, which is an exosome characterization marker, by flow cytometry device); FIG. 3E shows the graphical representation of the characterization of the exosome isolated from Pomegranate in the scope of the present invention (exosome diameter measurement graph);
[0056] FIG. 4A shows the graphical representation of the characterization of the exosome isolated from Leek in the scope of the present invention (flow cytometry graph measured by a control group, by flow cytometry device); FIG. 4B shows the graphical representation of the characterization of the exosome isolated from Leek in the scope of the present invention (flow cytometry graph measured by CD9 antibody, which is an exosome characterization marker, by flow cytometry device); FIG. 4C shows the graphical representation of the characterization of the exosome isolated from Leek in the scope of the present invention (flow cytometry graph measured by HSP70 antibody, which is an exosome characterization marker, by flow cytometry device); FIG. 4D shows the graphical representation of the characterization of the exosome isolated from Leek in the scope of the present invention (flow cytometry graph measured by CD63 antibody, which is an exosome characterization marker, by flow cytometry device); FIG. 4E shows the graphical representation of the characterization of the exosome isolated from Leek in the scope of the present invention (exosome diameter measurement graph);
[0057] FIG. 5A shows the graphical representation of the characterization of the exosome isolated from Horseradish in the scope of the present invention (flow cytometry graph measured by a control group, by flow cytometry device); FIG. 5B shows the graphical representation of the characterization of the exosome isolated from Horseradish in the scope of the present invention (flow cytometry graph measured by CD9 antibody, which is an exosome characterization marker, by flow cytometry device); FIG. 5C shows the graphical representation of the characterization of the exosome isolated from Horseradish in the scope of the present invention (flow cytometry graph measured by HSP70 antibody, which is an exosome characterization marker, by flow cytometry device); FIG. 5D shows the graphical representation of the characterization of the exosome isolated from Horseradish in the scope of the present invention (flow cytometry graph measured by CD63 antibody, which is an exosome characterization marker, by flow cytometry device); FIG. 5E shows the graphical representation of the characterization of the exosome isolated from Horseradish in the scope of the present invention (exosome diameter measurement graph);
[0058] FIG. 6A shows the graphical representation of the characterization of the exosome isolated from Ginger in the scope of the present invention (flow cytometry graph measured by a control group, by flow cytometry device); FIG. 6B shows the graphical representation of the characterization of the exosome isolated from Ginger in the scope of the present invention (flow cytometry graph measured by CD9 antibody, which is an exosome characterization marker, by flow cytometry device); FIG. 6C shows the graphical representation of the characterization of the exosome isolated from Ginger in the scope of the present invention (flow cytometry graph measured by HSP70 antibody, which is an exosome characterization marker, by flow cytometry device); FIG. 6D shows the graphical representation of the characterization of the exosome isolated from Ginger in the scope of the present invention (flow cytometry graph measured by CD63 antibody, which is an exosome characterization marker, by flow cytometry device); FIG. 6E shows the graphical representation of the characterization of the exosome isolated from Ginger in the scope of the present invention (exosome diameter measurement graph);
[0059] FIG. 7 shows the graphical representations of the effect of the Warty-Leaved Rhubarb exosome on white blood cells in the scope of the present invention (the graph of measurement of CD4 T helper lymphocytes. CD8 T cytotoxic lymphocytes, CD19 B lymphocytes and CD56 natural killer cells with the antibodies that are surface markers by flow cytometry device);
[0060] FIG. 8 shows the graphical representations of the effect of the Warty-Leaved Rhubarb exosome on IL2 activated white blood cells in the scope of the present invention (the graph of measurement of CD4 T helper lymphocytes, CD8 T cytotoxic lymphocytes, CD19 B lymphocytes and CD56 natural killer cells with the antibodies that are surface markers by flow cytometry device);
[0061] FIG. 9 shows the graphical representations of the effect of the Warty-Leaved Rhubarb exosome on PHA activated white blood cells in the scope of the present invention (the graph of measurement of CD4 T helper lymphocytes, CD8 T cytotoxic lymphocytes, CD19 B lymphocytes and CD56 natural killer cells with the antibodies that are surface markers by flow cytometry device);
[0062] FIG. 10 shows the graphical representations of the effect of the Warty-Leaved Rhubarb exosome on Mite Allergen activated white blood cells in the scope of the present invention (the graph of measurement of CD4 T helper lymphocytes, CD8 T cytotoxic lymphocytes, CD19 B lymphocytes and CD56 natural killer cells with the antibodies that are surface markers by flow cytometry device);
[0063] FIG. 11 shows the graphical representations of the effect of the Warty-Leaved Rhubarb exosome on Pollen Allergen activated white blood cells in the scope of the present invention (the graph of measurement of CD4 T helper lymphocytes, CD8 T cytotoxic lymphocytes, CD19 B lymphocytes and CD56 natural killer cells with the antibodies that are surface markers by flow cytometry device);
[0064] FIG. 12 shows the graphical representation of the effect of the Celery exosome on white blood cells in the scope of the present invention (the graph of measurement of CD4 T helper lymphocytes, CD8 T cytotoxic lymphocytes, CD19 B lymphocytes and CD56 natural killer cells with the antibodies that are surface markers by flow cytometry device);
[0065] FIG. 13 shows the graphical representations of the effect of the Celery exosome on IL2 activated white blood cells in the scope of the present invention (the graph of measurement of CD4 T helper lymphocytes, CD8 T cytotoxic lymphocytes, CD19 B lymphocytes and CD56 natural killer cells with the antibodies that are surface markers by flow cytometry device);
[0066] FIG. 14 shows the graphical representations of the effect of the Celery exosome on PHA activated white blood cells in the scope of the present invention (the graph of measurement of CD4 T helper lymphocytes, CD8 T cytotoxic lymphocytes, CD19 B lymphocytes and CD56 natural killer cells with the antibodies that are surface markers by flow cytometry device);
[0067] FIG. 15 shows the graphical representations of the effect of the Celery exosome on Mite Allergen activated white blood cells in the scope of the present invention (the graph of measurement of CD4 T helper lymphocytes, CD8 T cytotoxic lymphocytes, CD19 B lymphocytes and CD56 natural killer cells with the antibodies that are surface markers by flow cytometry device);
[0068] FIG. 16 shows the graphical representations of the effect of the Celery exosome on Pollen Allergen activated white blood cells in the scope of the present invention (the graph of measurement of CD4 T helper lymphocytes, CD8 T cytotoxic lymphocytes, CD19 B lymphocytes and CD56 natural killer cells with the antibodies that are surface markers by flow cytometry device);
[0069] FIG. 17 shows the graphical representation of the effect of the Pomegranate exosome on white blood cells in the scope of the present invention (the graph of measurement of CD4 T helper lymphocytes, CD8 T cytotoxic lymphocytes, CD19 B lymphocytes and CD56 natural killer cells with the antibodies that are surface markers by flow cytometry device);
[0070] FIG. 18 shows the graphical representations of the effect of the Pomegranate exosome on IL2 activated white blood cells in the scope of the present invention (the graph of measurement of CD4 T helper lymphocytes, CD8 T cytotoxic lymphocytes, CD19 B lymphocytes and CD56 natural killer cells with the antibodies that are surface markers by flow cytometry device);
[0071] FIG. 19 shows the graphical representations of the effect of the Pomegranate exosome on PHA activated white blood cells in the scope of the present invention (the graph of measurement of CD4 T helper lymphocytes, CD8 T cytotoxic lymphocytes, CD19 B lymphocytes and CD56 natural killer cells with the antibodies that are surface markers by flow cytometry device);
[0072] FIG. 20 shows the graphical representations of the effect of the Pomegranate exosome on Mite Allergen activated white blood cells in the scope of the present invention (the graph of measurement of CD4 T helper lymphocytes. CD8 T cytotoxic lymphocytes, CD19 B lymphocytes and CD56 natural killer cells with the antibodies that are surface markers by flow cytometry device);
[0073] FIG. 21 shows the graphical representations of the effect of the Pomegranate exosome on Pollen Allergen activated white blood cells in the scope of the present invention (the graph of measurement of CD4 T helper lymphocytes, CD8 T cytotoxic lymphocytes, CD19 B lymphocytes and CD56 natural killer cells with the antibodies that are surface markers by flow cytometry device);
[0074] FIG. 22 shows the graphical representation of the effect of the Leek exosome on white blood cells in the scope of the present invention (the graph of measurement of CD4 T helper lymphocytes, CD8 T cytotoxic lymphocytes, CD19 B lymphocytes and CD56 natural killer cells with the antibodies that are surface markers by flow cytometry device);
[0075] FIG. 23 shows the graphical representations of the effect of the Leek exosome on IL2 activated white blood cells in the scope of the present invention (the graph of measurement of CD4 T helper lymphocytes, CD8 T cytotoxic lymphocytes, CD19 B lymphocytes and CD56 natural killer cells with the antibodies that are surface markers by flow cytometry device);
[0076] FIG. 24 shows the graphical representations of the effect of the Leek exosome on PHA activated white blood cells in the scope of the present invention (the graph of measurement of CD4 T helper lymphocytes, CD8 T cytotoxic lymphocytes, CD19 B lymphocytes and CD56 natural killer cells with the antibodies that are surface markers by flow cytometry device);
[0077] FIG. 25 shows the graphical representations of the effect of the Leek exosome on Mite Allergen activated white blood cells in the scope of the present invention (the graph of measurement of CD4 T helper lymphocytes, CD8 T cytotoxic lymphocytes, CD19 B lymphocytes and CD56 natural killer cells with the antibodies that are surface markers by flow cytometry device);
[0078] FIG. 26 shows the graphical representations of the effect of the Leek exosome on Pollen Allergen activated white blood cells in the scope of the present invention (the graph of measurement of CD4 T helper lymphocytes, CD8 T cytotoxic lymphocytes, CD19 B lymphocytes and CD56 natural killer cells with the antibodies that are surface markers by flow cytometry device);
[0079] FIG. 27 shows the graphical representation of the effect of the Horseradish exosome on white blood cells in the scope of the present invention (the graph of measurement of CD4 T helper lymphocytes, CD8 T cytotoxic lymphocytes, CD19 B lymphocytes and CD56 natural killer cells with the antibodies that are surface markers by flow cytometry device);
[0080] FIG. 28 shows the graphical representations of the effect of the Horseradish exosome on IL2 activated white blood cells in the scope of the present invention (the graph of measurement of CD4 T helper lymphocytes, CD8 T cytotoxic lymphocytes, CD19 B lymphocytes and CD56 natural killer cells with the antibodies that are surface markers by flow cytometry device);
[0081] FIG. 29 shows the graphical representations of the effect of the Horseradish exosome on PHA activated white blood cells in the scope of the present invention (the graph of measurement of CD4 T helper lymphocytes, CD8 T cytotoxic lymphocytes, CD19 B lymphocytes and CD56 natural killer cells with the antibodies that are surface markers by flow cytometry device);
[0082] FIG. 30 shows the graphical representations of the effect of the Horseradish exosome on Mite Allergen activated white blood cells in the scope of the present invention (the graph of measurement of CD4 T helper lymphocytes, CD8 T cytotoxic lymphocytes, CD19 B lymphocytes and CD56 natural killer cells with the antibodies that are surface markers by flow cytometry device);
[0083] FIG. 31 shows the graphical representations of the effect of the Horseradish exosome on Pollen Allergen activated white blood cells in the scope of the present invention (the graph of measurement of CD4 T helper lymphocytes. CD8 T cytotoxic lymphocytes, CD19 B lymphocytes and CD56 natural killer cells with the antibodies that are surface markers by flow cytometry device);
[0084] FIG. 32 shows the graphical representation of the effect of the Ginger exosome on white blood cells in the scope of the present invention (the graph of measurement of CD4 T helper lymphocytes, CD8 T cytotoxic lymphocytes. CD19 B lymphocytes and CD56 natural killer cells with the antibodies that are surface markers by flow cytometry device);
[0085] FIG. 33 shows the graphical representations of the effect of the Ginger exosome on IL2 activated white blood cells in the scope of the present invention (the graph of measurement of CD4 T helper lymphocytes, CD8 T cytotoxic lymphocytes, CD19 B lymphocytes and CD56 natural killer cells with the antibodies that are surface markers by flow cytometry device);
[0086] FIG. 34 shows the graphical representations of the effect of the Ginger exosome on PHA activated white blood cells in the scope of the present invention (the graph of measurement of CD4 T helper lymphocytes, CD8 T cytotoxic lymphocytes, CD19 B lymphocytes and CD56 natural killer cells with the antibodies that are surface markers by flow cytometry device);
[0087] FIG. 35 shows the graphical representations of the effect of the Ginger exosome on Mite Allergen activated white blood cells in the scope of the present invention (the graph of measurement of CD4 T helper lymphocytes, CD8 T cytotoxic lymphocytes, CD19 B lymphocytes and CD56 natural killer cells with the antibodies that are surface markers by flow cytometry device); and
[0088] FIG. 36 shows the graphical representations of the effect of the Ginger exosome on Pollen Allergen activated white blood cells in the scope of the present invention (the graph of measurement of CD4 T helper lymphocytes. CD8 T cytotoxic lymphocytes, CD19 B lymphocytes and CD56 natural killer cells with the antibodies that are surface markers by flow cytometry device).
[0089] The subject matter of the present invention is to use plant exosomes isolated from plant lysates as immune system enhancers, silencers and modulators against diseases affecting immune system. The plant exosomes of the present invention are plant derived exosomes that enable prevention and treatment of diseases by activating, suppressing and modulating the immune system cells.
[0090] The effects of the plant exosomes of the present invention on the immune system are based on the effect on the division of immune system cells. The plant exosomes that activate the immune system stimulate the immune system cells enabling them to proliferate, thereby strengthening the immune response. The suppressive plant exosomes, on the other hand, make the immune system cells insensitive to division, thereby suppressing the immune system response. These effects of the exosomes discussed in the invention are shown in the data presented by changes in the surface proteins of the immune system cells to which they are applied (CD 8, CD 19 and CD 56).
[0091] In the scope of the invention, plant exosomes are used mainly in autoimmune diseases, and in cell, tissue, organ transplantations and in Graft Versus Host disease as immune system enhancers, suppressors or, if necessary, as modulators performing both of the first two functions.
[0092] Within the scope of the invention, the plant exosomes are obtained from at least one portion of the plant selected from the group consisting of the entire plant, fruit, leaf, seed, root or differentiated tissues like culture medium of the plant, stem cell, waste material, shell or phloem. Plant tissue culture is preferred as a source from which plant exosomes are obtained in order to produce exosomes at a concentration up to 5 times higher than the exosomes obtained from similar plants and to maintain the content and properties of the produced exosomes for a long time, thereby preventing them from being affected by the effects of the farm, harvesting, transport, etc.
[0093] The plant exosomes of the present invention are transfected to enable the cells in the plant tissue culture to produce proteins enhancing, suppressing or modulating the immunity system. Transfection is an external gene transfer to a cell. Transfection of the cells in plant culture can enable them to produce target proteins, and the exosomes secreted by these cells can thus contain these proteins.
[0094] Within the scope of the invention, the plant exosomes are isolated by an isolation method selected from the group consisting of isolation by two phase liquid system, graduated centrifuge, ultrafiltration, chromatographic methods, polymer based isolation and isolation by microbeads. Among them, the purest exosome isolation is achieved by isolation with two phase liquid system and therefore this isolation method is preferred within the scope of the present application.
[0095] The plant-derived exosomes of the present invention are used mainly in autoimmune diseases, and in cell, tissue, organ transplantations, in Graft Versus Host disease, and diseases where the immune system is affected such as rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus, sclerosis, Sjogren's syndrome, Type 1 Diabetes, allergic asthma, Wegener granulomatosis, Multiple Sclerosis, Crohn's disease, psoriasis, Graves' disease, Celiac Disease, alopecia areata (pelade), central nervous system vasculitis, Hashimoto's thyroiditis, myasthenia gravis, Goodpasture's syndrome, autoimmune hemolytic anemia, Guillan-Barre syndrome, polyarteritis nodosa, idiopathic thrombocytic purpura, temporal arteritis, primary biliary cirrhosis, Addison's Disease, ankylosing spondylitis, Reiter's syndrome, Takayazu's arteritis, and vitiligo as immune system enhancers, suppressors or, if necessary, modulators performing both of the first two functions. Plant-derived exosomes are administered orally, intranasally, intravenously, intramuscularly, intradermally, topically, intraperitoneally, and by injection for administering the effective dose of the selected plant exosomes to the patient to treat immune system-mediated diseases. They are also used for purposes of carrying immunomodulatory drugs, as an adjuvant for vaccination applications, and as a nutritional supplement for the purpose of modulating the immune system.
[0096] Method of isolation via two phase liquid system used for isolation of the plant exosomes used in the scope of the present invention comprises following steps: [0097] Disintegrating the plant, whose exosome will be isolated, by a blender to obtain the lysate thereof, [0098] Centrifuging at a stirring rate of 2,000 g to 10,000 g for 5-20 minutes for isolation of the exosomes from the plant lysate, [0099] Removing the particles of size 220 nm and above by filtration after centrifugation, [0100] Transferring the exosome-protein mixture obtained by centrifugation into a two phase liquid system containing PEG phase and DEX phase for separation thereof, [0101] Removing the nonexosomal proteins, cellular fat and other impurities from the exosomes by utilizing the chemical tendency of the PEG phase to the proteins and the DEX phase to the phospholipid structured membranes, [0102] Obtaining the isolated exosomes.
[0103] The present invention is for utilizing the effects of plant exosomes on the immune system as immune system enhancers, silencers and modulators against diseases. In the scope of the invention, the immunomodulatory effects of the plant exosomes are used mainly in autoimmune diseases, and in cell, tissue, organ transplantations and in diseases such as Graft Versus Host disease as immune system enhancers, suppressors or, if necessary, as modulators performing both of the first two functions. The effects of plant exosomes can vary according to the plant from which the exosome is isolated. While these can be the entire plant, fruit, leaf, seed and root, they may also be differentiated tissues like the plant's culture medium, stem cell, waste material, shell or phloem. The plant exosomes can be isolated by many methods such as isolation by two phase liquid system, graduated centrifuge, ultrafiltration, chromatographic methods, polymer based isolation and isolation by microbeads. The exosomes used in the study conducted within the scope of the invention are isolated from the plants disclosed in Table 1.
TABLE-US-00001 TABLE 1 The plants used for isolation of exosomes within the scope of the invention. Turkish English Latin Nar Pomegranate Punica granatum Pirasa Leek Allium ampeloprasum Kereviz Celery Apium graveolens Kara Turp Horseradish Radix Raphani nigri Eşgin Warty-Leaved Reun Ribes rhubarb Zencefil Ginger Zingiber officinale
[0104] The large size particles resulting from plant disintegration by centrifugation performed between 2,000 g and 10,000 g for 5-20 minutes for exosome isolation from plant lysate are intended not to cause any impurities in the dextran phase upon precipitating due to the centrifugation applied during the two phase separation process and their weights. In addition, it is ensured that the filter, which is used during the filtration process carried out for removing particles sized 220 nanometers and above, is not clogged. Exosomes are cleared of nonexosomal proteins, cellular fats and other impurities by utilizing the chemical tendency of the PEG phase to the proteins and the DEX phase to the phospholipid structured membranes in the two-phase liquid system. The DEX phase formed by means of the concentrations of the polymers that are used in the solution separate the exosomes.
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0105] The isolated exosomes are marked by the surface markers CD9, CD63 and HSP70 antibodies which are carried by the exosomes and the exosomes carrying these markers are measured by flow cytometry device. At the same time, the dimensions of the exosomes isolated with the Zeta Sizer device are also measured FIGS. 1A-E to 6A-E.
[0106] White blood cell isolation is performed to determine the effects of the plant exosomes on the blood cells. Blood introduced into a tube containing EDTA or a solution enabling blood clotting is mixed with PBS at a ratio of 1:1 by volume. It is carefully poured onto the ficoll solution in another tube without mixing the phases. The tube containing blood, PBS and ficoll is centrifuged for 15 minutes at approximately 3000 RPM. After centrifugation, the white intermediate phase containing the white blood cells between the upper plasma and the ficoll is withdrawn and transferred to a clean tube and it is washed by adding approximately 10 mL of PBS thereon. The cells are centrifuged again at 1500 RPM for 5 minutes. The cell pellet is removed and the white blood cell is cultured in the medium. These isolated white blood cells include cells which play a role in the immune system such as T cells, B cells, natural killer cells, and dendritic cells.
[0107] The cells grown in the medium are incubated with IL2, PHA, Mite Allergens and Pollen Allergens, thereby activating the immune system cells. The isolated plant exosomes are delivered to activated and unactivated blood cells. In order to demonstrate the effects of the plant exosomes on white blood cells, the percentages of the blood cells marked with CD4, CD8, CD19 and CD56 antibodies are measured by flow cytometry device. The effect of the Warty-Leaved Rhubarb exosomes on blood cells is shown in FIG. 7, the effect thereof on IL2 activated blood cells is shown in FIG. 8, the effect thereof on PHA activated blood cells is shown in FIG. 9, the effect thereof on Mite Allergen activated blood cells is shown in FIG. 10 and the effect thereof on Pollen Allergen activated blood cells is shown in FIG. 1. The effect of the Celery exosomes on blood cells is shown in FIG. 12, the effect thereof on IL2 activated blood cells is shown in FIG. 13, the effect thereof on PHA activated blood cells is shown in FIG. 14, the effect thereof on Mite Allergen activated blood cells is shown in FIG. 15 and the effect thereof on Pollen Allergen activated blood cells is shown in FIG. 16. The effect of the Pomegranate exosomes on blood cells is shown in FIG. 17, the effect thereof on IL2 activated blood cells is shown in FIG. 18, the effect thereof on PHA activated blood cells is shown in FIG. 19, the effect thereof on Mite Allergen activated blood cells is shown in FIG. 20 and the effect thereof on Pollen Allergen activated blood cells is shown in FIG. 21. The effect of the Leek exosomes on blood cells is shown in FIG. 22, the effect thereof on IL2 activated blood cells is shown in FIG. 23, the effect thereof on PHA activated blood cells is shown in FIG. 24, the effect thereof on Mite Allergen activated blood cells is shown in FIG. 25 and the effect thereof on Pollen Allergen activated blood cells is shown in FIG. 26. The effect of the Horseradish exosomes on blood cells is shown in FIG. 27, the effect thereof on IL2 activated blood cells is shown in FIG. 28, the effect thereof on PHA activated blood cells is shown in FIG. 29, the effect thereof on Mite Allergen activated blood cells is shown in FIG. and the effect thereof on Pollen Allergen activated blood cells is shown in FIG. 31. The effect of the Ginger exosomes on blood cells is shown in FIG. 32, the effect thereof on IL2 activated blood cells is shown in FIG. 33, the effect thereof on PHA activated blood cells is shown in FIG. 34, the effect thereof on Mite Allergen activated blood cells is shown in FIG. 35 and the effect thereof on Pollen Allergen activated blood cells is shown in FIG. 36.
[0108] Within the scope of the present invention, the advantages of using the effects of plant exosomes on the immune system as immune system enhancers, silencers and modulators against diseases can be listed as follows: [0109] The exosomes are entirely of plant origin, [0110] Activation is better than similar activators (IL2) and more successful than the substances obtained as similar chemicals, [0111] It is possible to obtain large amounts thereof inexpensively, [0112] The ratio of suppression of the activation of the blood cells is more successful than the substances obtained as similar chemicals, [0113] They do not have the toxic effects to the liver and the other organs that the other drugs have as they are completely of plant origin. [0114] They are a plant derived product which can be used in place of the stem cell treatment that is used for suppressing the immune system in tissue and organ transplantations, [0115] Since they do not contain contaminants of plant, animal or chemical origin, side effects due to the said contaminants are not encountered, [0116] The use of these exosomes replaces use of elements such as aluminum which is known to be harmful to body in case of overuse.
REFERENCES
[0117] [5]. Ludwig, A. K. and B. Giebel (2012). “Exosomes: small vesicles participating in intercellular communication.” Int J Biochem Cell Biol 44(1): 11-15. [0118] [6]. Iraci, N., T. Leonardi, F. Gessler, B. Vega and S. Pluchino (2016). “Focus on Extracellular Vesicles: Physiological Role and Signalling Properties of Extracellular Membrane Vesicles.” Int J Mol Sci 17(2): 171. [0119] [7]. Stegmayr, B. and G. Ronquist (1982). “Promotive effect on human sperm progressive motility by prostasomes.” Urol Res 10(5): 253-257. [0120] [8]. Gupta A, Punatar S, Mathew L, Kannan S, Khattry N. Cyclosporine Plus Methotrexate or Cyclosporine Plus Mycophenolate Mofetil as Graft Versus Host Disease Prophylaxis in Acute Leukemia Transplant: Comparison of Toxicity, Engraftment Kinetics and Transplant Outcome. Indian Journal of Hematology & Blood Transfusion 2016; 32(3):248-256. doi:10.1007/s12288-015-0577-3.
