Method for obtaining purified plant extract and exosomes having a particle size below 250 nanometers

Abstract

The invention relates to a method for the separation, purification, and industrial-scale production of exosomes from plant raw materials for use both as active and / or auxiliary substances in the food, cosmetic, and pharmaceutical industries and in scientific research conducted in the fields of molecular biology, biotechnology, food, agriculture, genetics, and medicine; and additionally for obtaining plant exosomes and purified and concentrated plant extracts while preserving their biological activity and without leaving chemical residues, characterized in that it comprises the process steps of: preparing the plant raw material by drying and grinding plant raw materials into powder form; performing filtration of the mixture of distilled water and plant raw material to remove coarse particles; performing low-speed centrifugation to allow the particles in the solution to settle and to achieve solid–liquid separation; cooling the mixture; performing a second filtration to remove remaining particles from the cooled and rested liquid; applying evaporation to remove excess water; performing ultrafiltration to remove impurity-forming particles; and performing homogenization to ensure uniform distribution of exosomes in the solution.

Description

[0001] DESCRIPTION

[0002] METHOD FOR OBTAINING PURIFIED PLANT EXTRACT AND EXOSOMES HAVING A PARTICLE SIZE BELOW 250 NANOMETERS

[0003] Technical Field

[0004] The invention relates to a method for the separation, purification, and industrial-scale production of exosomes from plant raw materials for use both as active and / or auxiliary substances in the food, cosmetic, and pharmaceutical industries and in scientific research conducted in the fields of molecular biology, biotechnology, food, agriculture, genetics, and medicine; and additionally, to a method for obtaining plant exosomes and purified and concentrated plant extracts while preserving their biological activity and without leaving chemical residues.

[0005] The invention particularly relates to a method that enables the production of extracts in a stable and high-bioavailability form without undergoing thermal and chemical degradation, by using process steps such as low-temperature vacuum evaporation, membrane filtration, and lyophilization, for obtaining plant exosomes as well as highly pure and biologically active plant extracts.

[0006] State of the Art

[0007] Exosomes are lipid bilayer vesicles secreted by cells and, when obtained from plant cells, generally have a diameter of approximately 50-250 nm. These nanovesicles, which play an important role in intercellular communication, carry biomolecules such as proteins, lipids, RNA, and DNA. Due to their biological functions, they are evaluated in various fields such as drug delivery systems, cellular regeneration, immune regulation, and use as biomarkers. They have significant potential particularly in the medical, biotechnology, pharmaceutical, cosmetic, and food industries. The biological advantages of exosomes are utilized in various applications such as the development of targeted drug delivery systems in cancer treatment, their use in the formulation of anti-aging skin care products, and the production of functional foods.

[0008] Exosomes can be isolated from biological fluids (blood, saliva, urine, milk, etc.) or from plant and animal cells. In the existing literature, various methods are available for exosome purification. These are classified as physical, chemical, and biological-based techniques.One of the commonly used methods today is ultracentrifugation and differential centrifugation. By applying centrifugation at different speeds, particles with different densities are separated. However, this method is not suitable for industrial scale use due to long processing times, high energy consumption, and low yield. Another commonly used method is ultrafiltration, in which particles are purified using selectively permeable membranes. However, it includes disadvantages such as membrane clogging and reduced efficiency.

[0009] Another technique used in exosome purification is chromatography methods. Although techniques such as affinity chromatography, ion-exchange chromatography, and gel filtration are effective in removing specific impurities, they are not suitable for large-scale production due to the requirement for expensive materials, long processing times, and low yield. In chemically based precipitation methods, exosomes are precipitated using polymer-based precipitation reagents or special solvents. However, disadvantages such as the formation of chemical impurities, deterioration of the biological integrity of the obtained exosomes, and low final product purity are observed as a result of this method.

[0010] In addition, methods such as immunoaffinity-based separation, density gradient centrifugation, and commercial purification kits are also included in the literature. However, these methods are generally suitable for research laboratories and are not appropriate for industrial-scale production. Among the common disadvantages of existing methods are high production costs, obtaining very low amounts of final product from very large amounts of raw material, degradation of the physical structural integrity of the obtained exosomes, and low product stability. Moreover, the inability of exosomes to remain intact for long periods under ambient conditions is one of the main factors limiting their use, especially in food and pharmaceutical applications. Therefore, there is a need for new methods that will enable the purification of exosomes on an industrial scale and their storage under appropriate conditions.

[0011] Jisu Kim and colleagues conducted a study entitled “Isolation and characterization of ginseng-derived exosome-like nanoparticles with sucrose cushioning followed by ultracentrifugation”. In this study, purification and characterization were performed to determine the efficacy of ginseng-derived exosome-like nanoparticles (GDEs). The GDEs were purified by sequential centrifugation, followed by ultracentrifugation using sucrose layers (68% and 27%). Size distribution, zeta potential, and morphology were analyzed by dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), and transmission electron microscopy (TEM). The total RNA and protein concentration of the GDEs was measured to determine quantity, and the stability of the freeze-dried form was tested for 90days. The results demonstrate that GDEs can be successfully isolated in a reproducible manner with high yield and purity. Spherical nanoparticles with an average diameter of 105.8 nm and a surface charge of -20.7 mV were obtained. According to NTA measurements, the particle concentration of the GDEs was determined to be 2.05 x 1013particles / mL. When protein and total RNA concentrations were analyzed, it was observed that the freeze-dried GDEs remained stable at room temperature for 60 days. This study presents a method that allows purification in a short time through sucrose-based double fractionation, demonstrating an approach suitable for the mass production of plant-derived exosome-like nanoparticles. In addition, it was shown that the stability of freeze-dried GDEs could be maintained for 60 days. Although a method was developed in this study for the isolation and stabilization of ginseng-derived exosome-like nanoparticles, there are some important limitations in terms of industrial-scale production. Firstly, the sequential centrifugation and ultracentrifugation methods used in the purification process are high-cost, time-consuming, and not suitable for large-scale production. In addition, sucrose-based purification methods may be insufficient to preserve the biological integrity of exosomes and may contain impurities. Although the stability of the obtained exosomes was maintained for 60 days, it is unclear whether this duration is sufficient for food and pharmaceutical applications. For long-term storage and industrial use, the stability of exosomes needs to be increased.

[0012] In the study entitled “Coffee-Derived Exosome-Like Nanoparticles: Are They the Secret Heroes?” conducted by Kantarcioglu Murat and colleagues, exosomes thought to be responsible for the health benefits of coffee were isolated. Exosomes were isolated from hot coffee beverages prepared from green and roasted coffee beans using size exclusion chromatography (SEC) and commercial kits. The exosomes were detected and characterized by transmission electron microscopy (TEM) and UV-Vis spectrophotometer analyses. It was determined that beverages prepared from green coffee contained more exosomes than roasted coffee, and that the kit-based isolation method was more efficient than the SEC method. In addition, the micro-RNA content of the isolated exosomes was characterized by microarray analyses. In this study, it was stated that coffee exosomes were identified for the first time and that these nanoparticles could exhibit therapeutic effects on chronic liver diseases. However, further experimental studies are required to verify this effect. Although the presence of coffee exosomes was demonstrated and their potential benefits were highlighted in the current study, sufficient information is not provided regarding the industrial-scale production, stability, degree of purity, and application areas of the isolated exosomes. The isolation methods used in the study (SEC and commercial kits), although successful at the laboratory scale, are not efficient, cost-effective, or sustainable for large-scale production. In addition, critical issues such as the chemical impurities of theisolated exosomes, whether they have a stable structure, and their suitability for long-term storage conditions were not evaluated. These deficiencies make it necessary to develop new methods in order for exosomes to be used reliably in industries such as food, pharmaceuticals, and cosmetics.

[0013] Another study conducted by Stephanie Andrade and colleagues entitled “Natural Compounds for Alzheimer’s Disease Therapy: A Systematic Review of Preclinic and Clinical Studies” aims to examine natural components that have so far been reported to provide significant benefits for this neurological disease and focuses on studies involving clinical trials. This study states that exosomes obtained particularly from plant, animal, and microbial sources may play an important role in the treatment of the disease and that the biological effects of these compounds are supported by clinical trials. However, exosomes obtained by current methods have various disadvantages. Existing exosome isolation and purification methods, especially conventional methods (ultracentrifugation, differential centrifugation, chromatography, etc.), encounter problems such as low yield, long processing times, high costs, and deterioration of the physical structure of the products. In addition, the stability of the obtained products is generally low, and their suitability for industrial production is particularly limited. The findings show that existing methods, especially in large-scale production, result in various impurities and low yield, and that the physical structural integrity of exosomes is degraded. Furthermore, it is not possible for the obtained exosomes to remain stable for long periods under ambient conditions. This situation constitutes a major obstacle, especially for use in sensitive fields such as the food and pharmaceutical industries.

[0014] Similarly, for obtaining plant extracts, aqueous extraction methods (maceration, infusion, decoction), extraction with alcohol or solvents (ethanol, methanol, etc.), supercritical CO2extraction (a method that leaves no solvent residue), and ultrasonic- and microwave-based extraction methods are used. Plant extracts are complex mixtures obtained from plants by various methods. As alternatives to traditional extraction methods, modern techniques such as ultrasound-assisted extraction have been developed. This method is more efficient compared to classical methods and increases the amount of bioactive substances and bioavailability. However, the purity level of the final product obtained and its suitability for industrial production pose problems in terms of use in the food and pharmaceutical industries. After extraction, techniques such as HPLC, TLC, HPTLC, MALDI-MS, and LC-MS are used for the purification and identification of bioactive compounds. Plant extraction methods are very important for obtaining bioactive compounds from plant materials. Traditional techniques such as maceration and Soxhlet extraction are widely used but arefrequently criticized due to high solvent consumption and long processing times. Due to the inefficiency of traditional methods and the impurity of the final products obtained, new methods have been sought; although modern techniques such as microwave-assisted, ultrasound-assisted, and supercritical fluid extraction show significant advantages compared to traditional methods, their suitability is still under discussion.

[0015] As a result, the existence of a need for an innovative method that eliminates the disadvantages present in the current technique and enables the separation, purification, and industrial-scale production of exosomes from plant raw materials, and the inadequacy of existing solutions, have made it necessary to carry out an improvement in the relevant technical field.

[0016] Brief Description of the Invention

[0017] The present invention relates to a method that meets the above-mentioned requirements, eliminates all disadvantages, and provides additional advantages, for the separation, purification, and industrial-scale production of exosomes from plant raw materials for use both as active and / or auxiliary substances in the food, cosmetic, and pharmaceutical industries and in scientific research conducted in the fields of molecular biology, biotechnology, food, agriculture, genetics, and medicine; and additionally, for obtaining plant exosomes and purified and concentrated plant extracts while preserving their biological activity and without leaving chemical residues.

[0018] Based on the known state of the art, the aim of the invention is to ensure that plant exosomes are purified entirely by physical methods without requiring the use of chemicals, and that, by providing biological integrity and high purity in the final product obtained, the biological activities of the exosomes are preserved, thereby making the final product suitable for the food, pharmaceutical, and cosmetic industries.

[0019] The aim of the invention is to enable large-scale production thanks to a modular structure suitable for industrial production, and thus to obtain an efficient system that can be easily adapted to different production volumes.

[0020] Another aim of the invention is to ensure the preservation of the biological structures of exosomes through low-speed centrifugation, and, unlike conventional methods, to obtain exosomes with high stability without damage to their chemical and physical structures.Another aim of the invention is to provide an environmentally and health-safe production process by adopting an environmentally friendly approach, eliminating the formation of chemical waste, and using entirely physical methods for sustainable production.

[0021] Another aim of the invention is to make the production process more economical due to the accessibility and low cost of the devices used, and, unlike commercial kits and complex methods, to provide a cost advantage in large-scale production.

[0022] Another aim of the invention is to obtain a faster and more cost-effective solution in terms of time and cost, since the production process is shorter and more efficient compared to conventional exosome purification methods.

[0023] Another aim of the invention is to ensure that products suitable for industrial use are obtained by obtaining exosomes with high purity while preserving their biological activity.

[0024] Another aim of the invention is to ensure that, by means of the developed method, directly highly purified and concentrated plant extracts can be obtained without the need to use chromatographic techniques.

[0025] Another aim of the invention is to ensure that, since the developed method is carried out using only water without requiring the use of any chemical substances compared to conventional extraction methods, natural and highly purified concentrated plant extract products can be obtained without the need to use high-cost devices that are not suitable for industrial production.

[0026] The structural and characteristic features of the invention and all its advantages will be understood more clearly through the detailed description given below; therefore, the evaluation should also be made by taking these detailed explanations into consideration.

[0027] Detailed Description of the Invention

[0028] In this detailed description, the method for the separation, purification, and industrial-scale production of exosomes from plant raw materials for use both as active and / or auxiliary substances in the food, cosmetic, and pharmaceutical industries and in scientific research conducted in the fields of molecular biology, biotechnology, food, agriculture, genetics, and medicine, as well as for obtaining plant exosomes and purified and concentrated plant extracts while preserving their biological activity and without leaving chemical residues,which is the subject of the invention, is described only as an example to facilitate better understanding of the subject and in a manner that does not create any limiting effect.

[0029] The method developed within the scope of the invention for the separation, purification, and industrial-scale production of exosomes from plant raw materials enables the obtained exosomes to be used as active substances and / or auxiliary, supportive substances in the food, cosmetic, and pharmaceutical industries. In addition, the obtained exosomes have the potential to be used in scientific research conducted in fields such as molecular biology, biotechnology, food, agriculture, genetics, and medicine.

[0030] The developed exosome purification method makes it possible to obtain a high amount of final product in a more stable form and with preserved physical structures compared to other exosome production methods reported in the literature. This system is suitable for the purification of exosomes from all plant sources due to its ease of use, suitability for industrial production, and operability. In addition, it has been proven that the obtained exosomes preserve their stable forms and activities at room temperature for a period of two years. The mentioned method includes the process steps of purifying mains water by distillation and preparing the plant raw materials by pulverizing them; mixing distilled water and the plant raw material and allowing it to rest; filtration to remove coarse particles; centrifugation to allow the particles in the solution to settle and to achieve solid-liquid separation; cooling; a second filtration to remove remaining particles from the cooled and rested liquid; resting; evaporation to remove excess water; ultrafiltration to remove impurity-forming particles; homogenization to ensure the uniform distribution of exosomes in the solution; freezing of the obtained homogeneous product; subjecting the frozen and rested product to lyophilization (freeze-drying) to convert it into powder form; and packaging of the obtained powder-form product. In the mentioned method, the solution is rested after each filtration step. Although ultracentrifugation is widely used, it generally results in lower particle recovery and higher protein purity. Density gradient separation and immunoaffinity capture methods have shown effectiveness in exosome isolation, with immunoaffinity capture standing out as a particularly efficient method. Size-exclusion chromatography has emerged as a promising method offering reproducibility, scalability, and cost effectiveness. In addition, ultrafiltration and precipitation methods have also been evaluated, and it has been determined that the ultracentrifugation method produces smaller and more homogeneous particles, and that these particles show increased effectiveness in protecting hypoxic cells. The choice of isolation method can have a significant effect on the concentration, purity, and size of exosomes and exosome-like RNA. This emphasizes the importance of carefully selecting the method based on research requirements. These findings demonstrate the necessity ofmaking a meticulous evaluation when selecting exosome isolation techniques for different applications. Since many of the existing methods involve high-cost, complex, and timeconsuming techniques, they are far from being suitable for industrial food production. Especially specific methods such as immunoaffinity capture require high sensitivity and specialized equipment that are difficult to adapt to large-scale production. Similarly, although methods such as size-exclusion chromatography are scalable, their economic feasibility remains limited in large-scale industrial applications. This situation reveals the need for simpler, faster, and lower-cost methods for exosome production intended for the food sector. When the production methods reported in the literature are examined and tested, the use of methods such as filtration, ultrafiltration, ultracentrifugation, differential centrifugation, chemical-based precipitation, chromatography, and commercial isolation kit methods has not been found suitable for industrial exosome purification. In particular, obtaining a product to be used in the food and pharmaceutical industries by treating it with chemicals through applications such as commercial exosome isolation kits, chromatographic methods, and chemical-based precipitation methods is not appropriate. The use of only ultrafiltration or ultracentrifugation alone does not allow the final product to be obtained in high yield without physical and chemical degradation. Due to the applied high pressure, high gravitational accelerations, and the effects of different physical forces, exosomes are fragmented, undergo aggregation, and their structures are degraded. With none of these methods can exosomes suitable for food consumption, purified at industrial scale, with preserved physical and chemical structure and high final product yield be obtained. Since the obtained products are not in stable form and are not dried, they cannot withstand sufficiently long periods even when stored at a temperature of -80°C. In the method that is the subject of the invention, exosomes can be obtained at the highest possible purity and in the highest final product yields by using only distilled water and a combination of physical methods without using any chemicals. By drying the purified final product through lyophilization, plant exosome products with a long shelf life and fully suitable for food are obtained.

[0031] For the exosome purification process, mains water is first distilled for 24 hours using a filtration-distillation system. The distilled water is collected in a storage tank. At the same time, plant raw materials are dried, ground, and converted into powder form.

[0032] The obtained distilled water and powdered plant raw material are transferred into a mixing and resting tank at a ratio of at least 1:1 and at most 100:1. Inside the tank, the temperature of the solution is preferably heated from 15°C up to 115°C, and the mixing speed is increased from 500 rpm up to 5000 rpm, and the mixing process is carried out for at least 6hours and at most 24 hours. After the mixing process is completed, the solution is rested under ambient conditions for at least 6 hours.

[0033] The rested solution is first passed through steel filters with preferably 5000 micron and then preferably 1000 micron pore diameters, and coarse particles are removed from the solution after filtration.

[0034] The filtered solution is collected in a resting tank and rested under ambient conditions for at least 12 hours.

[0035] The rested solution is subjected to differential centrifugation at forces of preferably first 1000 x g, then 3000 x g, and then 5000 x g, thereby allowing the particles in the solution to settle and solid-liquid separation to occur. This process lasts on average 24 hours. Thereafter, the solid phase is discarded and the liquid phase is collected in a cooling and resting tank.

[0036] The liquid product obtained after centrifugation is cooled in the cooling and resting tank until it falls below 24°C, preferably to between +4 and +24°C, and is rested under ambient conditions for 24 hours.

[0037] The rested product is sequentially passed through membrane filters with pore diameters of preferably 500 microns, 350 microns, and 200 microns within an average of 8 hours and collected in a resting tank. The product is rested in the resting tank for 24 hours under ambient conditions.

[0038] The rested product is subjected to an evaporation process for 10 hours to remove excess water from the solution.

[0039] Following the evaporation stage, the obtained solution is passed through membrane filters with pore diameters of preferably 150 microns, 100 microns, 50 microns, and 20 microns, respectively, for ultrafiltration, thereby ensuring the elimination of impurity-forming particles. Ultrafiltration enables the separation of exosome-sized particles using membrane technology. In existing applications, membranes with fixed pore diameters are generally used. Operation is carried out in a single stage, which prevents impurities from being completely eliminated. In addition, there is a high risk of clogging during the process, and membrane life may be short. In the method that is the subject of the invention, however, a multi-stage filtration process is employed. In the method of the invention, gradual membrane filtration processes are carried out from coarse particles to fine particles (5000 microns to 20microns). This ensures more precise removal of impurities in the solution. Since the product is transferred to resting tanks before each filtration step, the accumulation of particles on the membranes and the risk of clogging are minimized. In ultrafiltration, resting processes are generally not applied. In the method of the invention, performing resting after each filtration ensures that the particles remain stable in the solution and increases filtration efficiency. Ultrafiltration systems are generally at laboratory scale; however, the method of the invention has a modular structure that can be adapted to large-scale production.

[0040] The product obtained after filtration is collected in a resting tank and rested under ambient conditions for 12 hours.

[0041] In order to ensure uniform distribution of the exosomes contained in the rested product throughout the solution, homogenization is carried out under ambient conditions for 3 hours at a speed of preferably 2000 rpm using a mechanical homogenizer.

[0042] The obtained homogeneous product is transferred to freezing trays and frozen by shockfreezing at a temperature of preferably -60 / -80°C. The frozen product is stored at a temperature of -80°C for 48 hours prior to drying by lyophilization (freeze-drying).

[0043] The frozen and rested product is dried in a lyophilization machine preferably set at a temperature of 0°C for 48 hours until the moisture content is reduced to zero. After drying, the obtained powder product is collected from the trays, packaged, and stored at room temperature.

[0044] The plant exosome purification method that is the subject of the invention is an innovative solution that aims to eliminate the deficiencies of methods such as ultracentrifugation, ultrafiltration, chemical precipitation, use of commercial kits, and chromatography reported in the literature, and that is entirely physical, environmentally friendly, low-cost, and applicable at industrial scale. By completely eliminating the use of chemicals, it preserves the biological integrity of exosomes and provides high purity. Compared to conventional exosome isolation methods, the mentioned method offers a commercially applicable solution that supports sustainability and enables the production of large quantities of product.

[0045] In the method that is the subject of the invention, the combined use of entirely physical methods without requiring chemical usage ensures the acquisition of a natural product and the presentation of an environmentally friendly system. Compared to chemical-based precipitation or chromatography methods, high-purity plant exosomes can be obtained byeliminating impurities through the use of physical methods without requiring any chemical substances. Since no chemical residues are formed, the final product can be readily used in the food and pharmaceutical industries, and since no chemical waste is generated during production, an environmentally and sustainably friendly production process is achieved.

[0046] Due to the modular structure of the system suitable for industrial use and scalability, it can be easily adapted for production concepts of different sizes. The system has been developed for much larger-scale industrial plant exosome purification compared to conventional laboratorytype exosome isolation methods. Owing to modular filters, tanks, centrifuges, evaporators, chillers, and lyophilizer devices, the system can be scaled according to the designated production volume and efficient purification can be achieved. Due to its modular structure, it is suitable for exosome purification from all different plant sources.

[0047] In the method that is the subject of the invention, unlike the processes applied in conventional methods, the biological structures of exosomes are preserved due to the application of entirely physical methods without requiring high intensities. Multi-stage filtration and centrifugation processes at low intensities ensure the preservation of the chemical and physical structures of the obtained exosomes. Final products with ensured physical stability and high standardization have a long shelf life at room temperature. It is not possible for exosome-like products obtained by conventional methods in the literature to be stored at room temperature for long periods.

[0048] In the method that is the subject of the invention, a system that is much more advantageous in terms of cost and accessibility has been obtained thanks to the use of much more widely available equipment and materials compared to conventional methods. The equipment used in the mentioned method is much more easily obtainable and lower in cost compared to commercial kits or chromatography methods, thereby significantly increasing sustainability for industrial-scale production.

[0049] The invention provides an innovative method that enables not only the acquisition of plant exosomes but also the production of purified and concentrated plant extracts. Plant extracts can be used as active components in the food, cosmetic, and pharmaceutical industries. The mentioned method aims to produce plant extracts with high purity, without leaving chemical residues, and while preserving their biological activity. Plant extracts are complex biological mixtures containing natural chemical compounds, protein structures, extracellular vesicles, and various sugar components. Due to the diversity of these components, extracts contain a high level of impurities. When taken into the body, the bioavailability of the macromoleculesthey contain is limited, and access to smaller biological structures is restricted. However, as a result of reducing the particle size to below an average of 250 nm through the purification and concentration method of the extracts that are the subject of the invention, bioavailability at the cellular and tissue level increases. This process strengthens intercellular communication by enabling more effective passage of extracts through biological membranes and barriers and supports therapeutic activities. In addition, reducing particle size facilitates the delivery of extracts to the target tissue, offering more efficient use in biotechnological and pharmaceutical applications. The method that is the subject of the invention covers the production of highly pure and biologically active plant extracts in addition to plant exosomes. For the purification and concentration of plant extracts, techniques such as low-temperature vacuum evaporation, membrane filtration, and lyophilization are used, thereby ensuring that extracts are produced in a stable form with high bioavailability without undergoing thermal and chemical degradation. In the mentioned method, water-based extraction is applied to obtain highly purified extracts from plant raw materials. The liquid purified extract product obtained after extraction is concentrated by evaporation and freeze-drying (lyophilization) methods. Finally, the purified and concentrated extract is converted into powder form, packaged, and stabilized.

Claims

CLAIMS1. A method for the separation, purification, and industrial-scale production of exosomes from plant raw materials for use both as active and / or auxiliary substances in the food, cosmetic, and pharmaceutical industries and in scientific research conducted in the fields of molecular biology, biotechnology, food, agriculture, genetics, and medicine, and additionally for obtaining plant exosomes and purified and concentrated plant extracts while preserving their biological activity and without leaving chemical residues, characterized in that it comprises the process steps of: preparing the plant raw material by drying and grinding plant raw materials into powder form;performing filtration of the mixture of distilled water and plant raw material to remove coarse particles;performing low-speed centrifugation to allow the particles in the solution to settle and to achieve solid-liquid separation;cooling the mixture;performing a second filtration to remove remaining particles from the cooled and rested liquid;applying evaporation to remove excess water;performing ultrafiltration to remove impurity-forming particles;performing homogenization to ensure uniform distribution of exosomes in the solution.

2. The method according to Claim 1, characterized in that it comprises the process step of freezing the homogeneous product obtained after the homogenization stage.

3. The method according to Claim 2, characterized in that it comprises the process step of transferring the obtained homogeneous product to freezing trays and freezing it by shock-freezing at a temperature preferably of -60 / -80°C.

4. The method according to Claim 2, characterized in that it comprises the process step of storing the frozen product at a temperature of -80°C for 48 hours prior to drying by lyophilization (freeze-drying).

5. The method according to Claim 1, characterized in that it comprises the process step of converting the frozen and rested product into powder form by subjecting it to lyophilization (Freeze-drying).

6. The method according to Claim 5, characterized in that it comprises the process step of drying the frozen and rested product in a lyophilization machine preferably set at a temperature of 0°C for 48 hours until the moisture content is reduced to zero.

7. The method according to Claim 5, characterized in that it comprises the process step of collecting the powder product obtained after drying from the trays, packaging it, and storing it at room temperature.

8. The method according to Claim 1, characterized in that it comprises the process step of packaging the obtained powder-form product.

9. The method according to Claim 1, characterized in that it comprises the process step of distilling mains water for 24 hours using a filtration-distillation system and collecting the distilled water in a storage tank.

10. The method according to Claim 1, characterized in that it comprises the process step of mixing the obtained distilled water and the powdered plant raw material at a ratio of at least 1:1 and at most 100:1 and transferring them into a resting tank.

11. The method according to Claim 1, characterized in that it comprises the process step of heating the temperature of the solution in the resting tank preferably from 15°C up to 115°C and mixing it for at least 6 hours and at most 24 hours by increasing the mixing speed from 500 rpm up to 5000 rpm.

12. The method according to Claim 1, characterized in that it comprises the process step of resting the solution under ambient conditions for at least 6 hours after completion of the mixing.

13. The method according to Claim 1, characterized in that it comprises the process step of passing the rested solution first through steel filters with preferably 5000 micron pore diameter and then preferably 1000 micron pore diameter to remove coarse particles from the rested solution.

14. The method according to Claim 1, characterized in that it comprises the process step of collecting the filtered solution in a resting tank and resting it under ambient conditions for at least 12 hours.

15. The method according to Claim 1, characterized in that it comprises the process step of subjecting the rested solution to differential centrifugation for an average of 24 hours at forces of preferably first 1000 x g, then 3000 x g, and then 5000 x g, in order to allow the particles in the solution to settle and to achieve solid-liquid separation.

16. The method according to Claim 1, characterized in that it comprises the process step of cooling the liquid product obtained after centrifugation in a cooling and resting tank until it falls below 24°C, preferably to between +4 and +24°C, and resting it under ambient conditions for 24 hours.

17. The method according to Claim 1, characterized in that it comprises the process step of sequentially passing the rested product through membrane filters with pore diameters of preferably 500 microns, 350 microns, and 200 microns within an average of 8 hours.

18. The method according to Claim 1, characterized in that it comprises the process step of collecting the product passed through the filters in a resting tank and resting it under ambient conditions preferably for 24 hours.

19. The method according to Claim 1, characterized in that it comprises the process step of applying evaporation to the rested product for 10 hours in order to remove excess water from the solution.

20. The method according to Claim 1, characterized in that it comprises the process step of, after the evaporation stage, passing the obtained solution through membrane filters with pore diameters of preferably 150 microns, 100 microns, 50 microns, and 20 microns, respectively, for ultrafiltration in order to eliminate impurity-forming particles.

21. The method according to Claim 1, characterized in that it comprises the process step of collecting the product obtained after ultrafiltration in a resting tank and resting it under ambient conditions for 12 hours.

22. The method according to Claim 1, characterized in that it comprises the process step of carrying out homogenization under ambient conditions for 3 hours at a speed of preferably 2000 rpm using a mechanical homogenizer in order to ensure uniform distribution of the exosomes contained in the rested product throughout the solution.

23. The method according to Claim 1, characterized in that it comprises the process step of performing resting after each filtration in order to ensure that the particles remain stable in the solution and to increase filtration efficiency.

24. The method according to Claim 1, characterized in that it comprises the process step of applying water-based extraction to obtain highly purified extracts from plant raw materials.

25. The method according to Claim 24, characterized in that it comprises the process step of concentrating the liquid purified extract product obtained after extraction by evaporation and lyophilization (Freeze-drying) methods.

26. The method according to Claim 25, characterized in that it comprises the process step of converting the purified and concentrated extract into powder form and packaging it.

27. The method according to Claim 1, characterized in that it comprises the process step of reducing the particle size to below an average of 250 nm in order to increase bioavailability at the cellular and tissue level.