Saffron exosome as well as preparation method and application thereof

Abstract

The application belongs to the technical field of exosomes, and particularly relates to a saffron exosome and a preparation method and application thereof. The preparation method comprises the following steps: collecting saffron stigmas and petals, washing and drying, crushing to 80-100 meshes to obtain saffron powder; soaking the saffron powder in a specific solvent according to a solid-liquid ratio of 1:6-10, and then performing ultrasonic extraction to obtain an extraction liquid; wherein the ultrasonic power is set to 200-300 W, the time is 30-60 minutes, and the temperature is controlled at 4-8 DEG C; the specific solvent formula is as follows: 0.005-0.01 g of potassium nitrate or 0.01-0.02 g of potassium sulfate is added to each liter of PBS buffer solution; the extraction liquid is first centrifuged at a speed of 8000-10000 g to remove impurity precipitates, and the supernatant is filtered through a 100-120 kDa ultrafiltration membrane, and the filtrate is collected; the filtrate is subjected to ultracentrifugation at a speed of 100000-110000 g for 70-90 minutes to obtain exosome precipitates, which are saffron exosomes. The application can stimulate the release of exosomes, stabilize and retain saffron exosomes, and finally significantly improve the extraction efficiency of saffron exosomes, so that the best activity is achieved.

Description

Technical Field

[0001] This invention belongs to the field of exosome technology, specifically relating to a saffron exosome, its preparation method, and its application. Background Technology

[0002] Exosomes are nanoscale vesicles secreted by cells and play a crucial role in living organisms. Based on current research, the physiological functions of exosomes are mainly manifested in the following aspects:

[0003] (1) Signal transduction: Exosomes can carry ligands, receptors or small RNA molecules to regulate signaling pathways and thus regulate physiological functions.

[0004] (2) Immunomodulation: Some exosomes can induce T cell immune responses and inhibit the secretion of inflammatory factors such as IL-10 and TGF-β, thereby inhibiting inflammation and regulating immunity.

[0005] (3) Tissue repair: Exosomes can accumulate factors that promote skin growth, thereby promoting angiogenesis, repairing skin damage, and anti-aging.

[0006] For the reasons mentioned above, exosomes are widely used in many biopharmaceutical research projects, such as mesenchymal stem cell exosomes, neural stem cell exosomes, and blood cell exosomes. Many methods exist for exosome extraction, including ultracentrifugation, density gradient centrifugation, and ultrafiltration. However, these methods primarily isolate exosomes from cell secretions.

[0007] Saffron, a valuable medicinal plant, has its extracts widely used in medicine, cosmetics, and other fields. However, research on saffron exosomes is relatively limited, particularly regarding shortcomings in extraction processes. Studies have found that traditional extraction methods often result in low extraction efficiency and difficulty in obtaining high-purity exosomes, thus restricting their application. Summary of the Invention

[0008] To address the aforementioned technical problems, this invention provides saffron exosomes, their preparation method, and applications. High-purity saffron exosomes with sizes between 30 nm and 150 nm can be extracted, laying the foundation for widespread application.

[0009] This invention provides a method for preparing saffron exosomes, comprising the following steps:

[0010] The stigmas and petals of saffron are collected, washed, dried, and then pulverized to 80-100 mesh to obtain saffron powder.

[0011] Saffron powder was soaked in a specific solvent at a material-to-liquid ratio of 1:6 to 10, and then subjected to ultrasonic extraction to obtain an extract. The ultrasonic power was set to 200W to 300W, the time to 30 to 60 minutes, and the temperature was controlled at 4℃ to 8℃. The specific solvent formula was as follows: 0.005g to 0.01g of potassium nitrate or 0.01g to 0.02g of potassium sulfate per liter of PBS buffer.

[0012] The extract was centrifuged at 8000g~10000g to remove impurities and precipitates. The supernatant was filtered through a 100kDa~120kDa ultrafiltration membrane, and the filtrate was collected. The filtrate was then centrifuged at 100000g~110000g for 70 minutes~90 minutes to obtain exosome precipitates, which are saffron exosomes.

[0013] Preferably, in the above method for preparing saffron exosomes, the mass of potassium nitrate added per liter of PBS buffer is 0.007 g, or the mass of potassium sulfate added per liter of PBS buffer is 0.015 g.

[0014] Preferably, in the above-mentioned method for preparing saffron exosomes, the PBS buffer formulation per liter is as follows: 8g sodium chloride, 0.2g potassium chloride, 2.9g disodium hydrogen phosphate, 0.24g potassium dihydrogen phosphate, and deionized water to make up to 1L; the pH of the PBS buffer is 7.2~7.4.

[0015] Preferably, in the above-mentioned method for preparing saffron exosomes, the exosome precipitate is freeze-dried to obtain exosome lyophilized powder.

[0016] Preferably, the above-mentioned method for preparing saffron exosomes further includes a biological modification step of the exosomes, the method being as follows: adding 0.5% to 1% of a protease equivalent to the mass of the extract to the extract, and enzymatically hydrolyzing at 40℃ to 50℃ for 1 to 2 hours to obtain the enzymatic hydrolysis product;

[0017] After obtaining the enzymatic hydrolysis product, the step of centrifuging at a speed of 8000g~10000g to remove impurities and precipitates is then performed.

[0018] Preferably, in the above-mentioned method for preparing saffron exosomes, the protease is trypsin or papain.

[0019] Preferably, in the above-mentioned method for preparing saffron exosomes, the washing and drying conditions are: washing with tap water and drying at 0°C.

[0020] This invention provides a saffron exosome prepared by the preparation method described above.

[0021] Application of saffron exosomes in the preparation of antibacterial agents and / or antioxidants.

[0022] Preferably, the antibacterial activity refers to antibacterial activity against Escherichia coli, Staphylococcus aureus, and drug-resistant Staphylococcus aureus;

[0023] The term "antioxidant" refers to the removal of free radicals.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] This invention is the first to discover that the solvent has a significant impact on the extraction efficiency of exosomes, and specifies a particular solvent formulation as 1L of PBS buffer + 0.005g~0.01g of potassium nitrate, or 1L of PBS buffer + 0.01g~0.02g of potassium sulfate. Compared with deionized water and conventional PBS buffer, this specific solvent can stimulate the release of exosomes, stabilize and retain saffron exosomes, and ultimately significantly improve the extraction efficiency and purity of saffron exosomes, achieving the best extraction effect.

[0026] To further improve purity, techniques such as affinity chromatography and dual-dendritic magnetic microfibers can be combined to achieve efficient separation by utilizing the specific binding of exosome surface markers with the chromatographic medium.

[0027] This invention also provides a method for the biomodification of exosomes. The main components of saffron exosomes include nucleic acids, proteins, and carotenoids and other secondary metabolites. By adding 0.5% to 1% of a protease equivalent to the mass of the extract to the extract, the proteins in the saffron exosomes are degraded into peptides with smaller molecular weights, thereby changing the composition of the exosomes. This achieves the function of disrupting the bacterial membrane structure and disrupting the membrane potential, thereby increasing the sensitivity to bacteria and producing a stronger antibacterial effect. Attached Figure Description

[0028] Figure 1 This is a schematic diagram illustrating the inventive concept of the present invention. Detailed Implementation

[0029] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments and accompanying drawings.

[0030] Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0031] The inventive concept of this invention refers to... Figure 1 It mainly includes the following:

[0032] (1) Summary of existing technical problems:

[0033] Existing technologies such as ultracentrifugation, density gradient centrifugation, and ultrafiltration are mainly used to separate exosomes from cell secretions and are mostly suitable for the extraction of exosomes from animal-derived cells such as mesenchymal stem cells.

[0034] (2) Characteristic analysis of saffron as a source of exosome extraction:

[0035] Saffron is a plant, and its cell structure differs from that of animal cells. For example, saffron cells contain special components such as cell walls and cuticles, which hinder the secretion of exosomes outside the cell, thus affecting the extraction efficiency of exosomes.

[0036] (3) Design of the technical solution of the present invention

[0037] To overcome the above-mentioned technical bottlenecks, the present invention provides a method for preparing saffron exosomes, comprising the following steps:

[0038] Step 1, Raw material processing: Select high-quality saffron stigmas and petals, wash and dry them, and then pulverize them to 80-100 mesh to obtain saffron powder, in order to increase the efficiency of subsequent extraction.

[0039] High-quality saffron refers to saffron stigmas and petals that are undamaged, disease-free, and normally developed.

[0040] The cleaning and drying can be carried out using conventional cleaning and drying methods in the field. The embodiments of the present invention provide exemplary solutions and should not be construed as limiting the present invention.

[0041] Step 2, Extraction Process:

[0042] Saffron powder was soaked in a specific solvent at a material-to-liquid ratio of 1:6 to 10, and then subjected to ultrasonic extraction to obtain an extract. The ultrasonic power was set to 200W to 300W, the time to 30 to 60 minutes, and the ultrasonic temperature was controlled at 4℃ to 8℃.

[0043] On the one hand, ultrasonic extraction can effectively disrupt cell structure and release exosomes. This invention discovers that different ultrasonic powers, times, and temperatures affect the extraction efficiency of exosomes, and limits the appropriate ultrasonic power, time, and temperature to achieve the best extraction effect.

[0044] On the other hand, this invention is the first to discover that the solvent has a significant impact on the extraction efficiency of exosomes, and specifies a particular solvent formulation as 1L of PBS buffer + 0.005g~0.01g of potassium nitrate, or 1L of PBS buffer + 0.01g~0.02g of potassium sulfate. Compared with deionized water and conventional PBS buffer, this specific solvent can stimulate the release of exosomes and stabilize and retain saffron exosomes, ultimately significantly improving the purity of saffron exosomes and thus improving the extraction efficiency.

[0045] Step 3, Purification: The extract is centrifuged at 8000g~10000g to remove impurities and precipitates. The supernatant is filtered through a 100kDa~120kDa ultrafiltration membrane, and the filtrate is collected. The filtrate is then ultracentrifuged at 100000g~110000g for 70 minutes~90 minutes to obtain exosome precipitate, which is saffron exosomes. The exosome precipitate contains a high concentration of saffron exosomes. This step, through multiple steps and centrifugation at different speeds, removes impurities step by step, improving the purity of saffron exosomes.

[0046] Step 4, Drying and Preservation: The purified exosomes are preserved by freeze-drying at -20℃ to ensure their stability and activity during long-term storage.

[0047] In addition, to further improve purity, the following advanced purification step is added between step 3 and step 4: combining affinity chromatography and other chromatography techniques to achieve efficient separation by utilizing the specific binding of specific markers on the surface of exosomes with the chromatographic medium.

[0048] Alternatively, between steps 3 and 4, the following advanced purification step can be added: The exosome precipitate is resuspended and mixed with double-dendritic magnetic microfibrils (fDDMMs, described in "Chang W, Cai Y, Sun J, et al. Surface DoubleDendritic Magnetic Microfibrils for Rapid Isolation and Proteomic Profiling of Extracellular Vesicles from Microliters of Biofluids[J]. ACS Nano, 2025, 19(9):9196-9206.DOI:10.1021 / acsnano.4c18711") to separate and capture the exosomes. This material can rapidly separate and capture extracellular vesicles (EVs) from microliter-sized biofluids and can be directly used for proteomics analysis. The use of fDDMMs not only improves separation efficiency but also maintains the integrity and bioactivity of exosomes, providing high-quality samples for subsequent analysis and applications. The specific operation for deep purification using fDDMMs technology is as follows: resuspend the exosome precipitate, mix the resulting exosome solution with fDDMMs at a mass ratio of 10:1, gently shake for 5 to 10 minutes at room temperature, then use a magnet to separate the fDDMMs containing exosomes, wash several times with washing solution to remove unbound impurities, and finally elute the exosomes from the fDDMMs by elution to obtain high-purity saffron exosomes.

[0049] Based on the same inventive concept, this invention also provides a method for the biomodification of exosomes. The main components of saffron exosomes include nucleic acids, proteins, and secondary metabolites such as carotenoids. By adding 0.5% to 1% of a protease equivalent to the mass of the extract to the extract, the proteins in the saffron exosomes are degraded into polypeptides with smaller molecular weights, thereby changing the composition of the exosomes. This achieves the function of disrupting the bacterial membrane structure and disturbing the membrane potential, thereby increasing the sensitivity to bacteria and producing a stronger antibacterial effect.

[0050] The following are specific embodiments of the present invention.

[0051] Example 1

[0052] A method for preparing saffron exosomes includes the following steps:

[0053] Step 1, Raw material processing: Collect high-quality saffron stigmas and petals, wash them with tap water, dry them at 0℃, and then pulverize them to 80 mesh to obtain saffron powder, in order to increase the efficiency of subsequent extraction.

[0054] High-quality saffron refers to saffron that is undamaged, disease-free, and normally developed.

[0055] Step 2, Extraction Process:

[0056] Saffron powder was soaked in a specific solvent at a material-to-liquid ratio of 1:6, and then subjected to ultrasonic extraction to obtain an extract. The ultrasonic power was set to 200W, the time to 30 minutes, and the temperature was controlled at 4℃.

[0057] The specific solvent formulation is: 0.005 g potassium nitrate per liter of PBS buffer. The PBS buffer formulation per liter is as follows: 8 g sodium chloride, 0.2 g potassium chloride, 2.9 g disodium hydrogen phosphate, 0.24 g potassium dihydrogen phosphate, and deionized water to make up to 1 L; the pH of the PBS buffer is 7.2.

[0058] Step 3, Purification: The extract was centrifuged at 10000g for 10 minutes to remove impurities and precipitate. The supernatant was then filtered through a 100kDa ultrafiltration membrane, and the filtrate was collected. The filtrate was then centrifuged at 100000g for 70 minutes to obtain exosome precipitate. The exosome precipitate was purified exosomes containing a high concentration of saffron exosomes.

[0059] Step 4, Drying and Preservation: The purified exosomes are preserved by freeze-drying at -20℃ to ensure their stability and activity during long-term storage.

[0060] Example 2

[0061] A method for preparing saffron exosomes includes the following steps:

[0062] Step 1, Raw material processing: Collect high-quality saffron stigmas and petals, wash them with tap water, dry them at 0℃, and then pulverize them to 80 mesh to obtain saffron powder, in order to increase the efficiency of subsequent extraction.

[0063] High-quality saffron refers to saffron that is undamaged, disease-free, and normally developed.

[0064] Step 2, Extraction Process:

[0065] Saffron powder was soaked in a specific solvent at a material-to-liquid ratio of 1:6, and then subjected to ultrasonic extraction to obtain an extract. The ultrasonic power was set to 300W, the time to 30 minutes, and the temperature was controlled at 4℃.

[0066] The specific solvent formulation is: 0.005 g potassium nitrate per liter of PBS buffer. The PBS buffer formulation per liter is as follows: 8 g sodium chloride, 0.2 g potassium chloride, 2.9 g disodium hydrogen phosphate, 0.24 g potassium dihydrogen phosphate, and deionized water to make up to 1 L; the pH of the PBS buffer is 7.2.

[0067] Step 3, Purification: The extract was centrifuged at 10000g for 10 minutes to remove impurities and precipitate. The supernatant was then filtered through a 100kDa ultrafiltration membrane, and the filtrate was collected. The filtrate was then centrifuged at 100000g for 70 minutes to obtain exosome precipitate. The exosome precipitate was purified exosomes containing a high concentration of saffron exosomes.

[0068] Step 4, Drying and Preservation: The purified exosomes are preserved by freeze-drying at -20℃ to ensure their stability and activity during long-term storage.

[0069] Example 3

[0070] A method for preparing saffron exosomes includes the following steps:

[0071] Step 1, Raw material processing: Collect high-quality saffron stigmas and petals, wash them with tap water, dry them at 0℃, and then pulverize them to 80 mesh to obtain saffron powder, in order to increase the efficiency of subsequent extraction.

[0072] High-quality saffron refers to saffron that is undamaged, disease-free, and normally developed.

[0073] Step 2, Extraction Process:

[0074] Saffron powder was soaked in a specific solvent at a material-to-liquid ratio of 1:6, and then subjected to ultrasonic extraction to obtain an extract. The ultrasonic power was set to 200W, the time to 60 minutes, and the temperature was controlled at 4℃.

[0075] The specific solvent formulation is: 0.005 g potassium nitrate per liter of PBS buffer. The PBS buffer formulation per liter is as follows: 8 g sodium chloride, 0.2 g potassium chloride, 2.9 g disodium hydrogen phosphate, 0.24 g potassium dihydrogen phosphate, and deionized water to make up to 1 L; the pH of the PBS buffer is 7.2.

[0076] Step 3, Purification: The extract was centrifuged at 10000g for 10 minutes to remove impurities and precipitate. The supernatant was then filtered through a 100kDa ultrafiltration membrane, and the filtrate was collected. The filtrate was then centrifuged at 100000g for 70 minutes to obtain exosome precipitate. The exosome precipitate was purified exosomes containing a high concentration of saffron exosomes.

[0077] Step 4, Drying and Preservation: The purified exosomes are preserved by freeze-drying at -20℃ to ensure their stability and activity during long-term storage.

[0078] Example 4

[0079] A method for preparing saffron exosomes includes the following steps:

[0080] Step 1, Raw material processing: Collect high-quality saffron stigmas and petals, wash them with tap water, dry them at 0℃, and then pulverize them to 80 mesh to obtain saffron powder, in order to increase the efficiency of subsequent extraction.

[0081] High-quality saffron refers to saffron that is undamaged, disease-free, and normally developed.

[0082] Step 2, Extraction Process:

[0083] Saffron powder was soaked in a specific solvent at a material-to-liquid ratio of 1:6, and then subjected to ultrasonic extraction to obtain an extract. The ultrasonic power was set to 200W, the time to 30 minutes, and the temperature was controlled at 8℃.

[0084] The specific solvent formulation is: 0.005 g potassium nitrate per liter of PBS buffer. The PBS buffer formulation per liter is as follows: 8 g sodium chloride, 0.2 g potassium chloride, 2.9 g disodium hydrogen phosphate, 0.24 g potassium dihydrogen phosphate, and deionized water to make up to 1 L; the pH of the PBS buffer is 7.2.

[0085] Step 3, Purification: The extract was centrifuged at 10000g for 10 minutes to remove impurities and precipitate. The supernatant was then filtered through a 100kDa ultrafiltration membrane, and the filtrate was collected. The filtrate was then centrifuged at 100000g for 70 minutes to obtain exosome precipitate. The exosome precipitate was purified exosomes containing a high concentration of saffron exosomes.

[0086] Step 4, Drying and Preservation: The purified exosomes are preserved by freeze-drying at -20℃ to ensure their stability and activity during long-term storage.

[0087] Example 5

[0088] A method for preparing saffron exosomes includes the following steps:

[0089] Step 1, Raw material processing: Collect high-quality saffron stigmas and petals, wash them with tap water, dry them at 0℃, and then pulverize them to 80 mesh to obtain saffron powder, in order to increase the efficiency of subsequent extraction.

[0090] High-quality saffron refers to saffron that is undamaged, disease-free, and normally developed.

[0091] Step 2, Extraction Process:

[0092] Saffron powder was soaked in a specific solvent at a material-to-liquid ratio of 1:6, and then subjected to ultrasonic extraction to obtain an extract. The ultrasonic power was set to 200W, the time to 30 minutes, and the temperature was controlled at 4℃.

[0093] The specific solvent formulation is: 0.01g potassium nitrate per liter of PBS buffer. The PBS buffer formulation per liter is as follows: 8g sodium chloride, 0.2g potassium chloride, 2.9g disodium hydrogen phosphate, 0.24g potassium dihydrogen phosphate, and deionized water to make up to 1L; the pH of the PBS buffer is 7.2.

[0094] Step 3, Purification: The extract was centrifuged at 10000g for 10 minutes to remove impurities and precipitate. The supernatant was then filtered through a 100kDa ultrafiltration membrane, and the filtrate was collected. The filtrate was then centrifuged at 100000g for 70 minutes to obtain exosome precipitate. The exosome precipitate was purified exosomes containing a high concentration of saffron exosomes.

[0095] Step 4, Drying and Preservation: The purified exosomes are preserved by freeze-drying at -20℃ to ensure their stability and activity during long-term storage.

[0096] Example 6

[0097] A method for preparing saffron exosomes includes the following steps:

[0098] Step 1, Raw material processing: Collect high-quality saffron stigmas and petals, wash them with tap water, dry them at 0℃, and then pulverize them to 80 mesh to obtain saffron powder, in order to increase the efficiency of subsequent extraction.

[0099] High-quality saffron refers to saffron that is undamaged, disease-free, and normally developed.

[0100] Step 2, Extraction Process:

[0101] Saffron powder was soaked in a specific solvent at a material-to-liquid ratio of 1:6, and then subjected to ultrasonic extraction to obtain an extract. The ultrasonic power was set to 200W, the time to 30 minutes, and the temperature was controlled at 4℃.

[0102] The specific solvent formulation is: 0.007 g potassium nitrate per liter of PBS buffer. The PBS buffer formulation per liter is as follows: 8 g sodium chloride, 0.2 g potassium chloride, 2.9 g disodium hydrogen phosphate, 0.24 g potassium dihydrogen phosphate, and deionized water to make up to 1 L; the pH of the PBS buffer is 7.2.

[0103] Step 3, Purification: The extract was centrifuged at 10000g for 10 minutes to remove impurities and precipitate. The supernatant was then filtered through a 100kDa ultrafiltration membrane, and the filtrate was collected. The filtrate was then centrifuged at 100000g for 70 minutes to obtain exosome precipitate. The exosome precipitate was purified exosomes containing a high concentration of saffron exosomes.

[0104] Step 4, Drying and Preservation: The purified exosomes are preserved by freeze-drying at -20℃ to ensure their stability and activity during long-term storage.

[0105] Example 7

[0106] A method for preparing saffron exosomes includes the following steps:

[0107] Step 1, Raw material processing: Collect high-quality saffron stigmas and petals, wash them with tap water, dry them at 0℃, and then pulverize them to 80 mesh to obtain saffron powder, in order to increase the efficiency of subsequent extraction.

[0108] High-quality saffron refers to saffron that is undamaged, disease-free, and normally developed.

[0109] Step 2, Extraction Process:

[0110] Saffron powder was soaked in a specific solvent at a material-to-liquid ratio of 1:6, and then subjected to ultrasonic extraction to obtain an extract. The ultrasonic power was set to 200W, the time to 30 minutes, and the temperature was controlled at 4℃.

[0111] The specific solvent formulation is: 0.01g potassium sulfate per liter of PBS buffer. The PBS buffer formulation per liter is as follows: 8g sodium chloride, 0.2g potassium chloride, 2.9g disodium hydrogen phosphate, 0.24g potassium dihydrogen phosphate, and deionized water to make up to 1L; the pH of the PBS buffer is 7.2.

[0112] Step 3, Purification: The extract was centrifuged at 10000g for 10 minutes to remove impurities and precipitate. The supernatant was then filtered through a 100kDa ultrafiltration membrane, and the filtrate was collected. The filtrate was then centrifuged at 100000g for 70 minutes to obtain exosome precipitate. The exosome precipitate was purified exosomes containing a high concentration of saffron exosomes.

[0113] Step 4, Drying and Preservation: The purified exosomes are preserved by freeze-drying at -20℃ to ensure their stability and activity during long-term storage.

[0114] Example 8

[0115] A method for preparing saffron exosomes includes the following steps:

[0116] Step 1, Raw material processing: Collect high-quality saffron stigmas and petals, wash them with tap water, dry them at 0℃, and then pulverize them to 80 mesh to obtain saffron powder, in order to increase the efficiency of subsequent extraction.

[0117] High-quality saffron refers to saffron that is undamaged, disease-free, and normally developed.

[0118] Step 2, Extraction Process:

[0119] Saffron powder was soaked in a specific solvent at a material-to-liquid ratio of 1:6, and then subjected to ultrasonic extraction to obtain an extract. The ultrasonic power was set to 200W, the time to 30 minutes, and the temperature was controlled at 4℃.

[0120] The specific solvent formulation is: 0.02g potassium sulfate per liter of PBS buffer. The PBS buffer formulation per liter is as follows: 8g sodium chloride, 0.2g potassium chloride, 2.9g disodium hydrogen phosphate, 0.24g potassium dihydrogen phosphate, and deionized water to make up to 1L; the pH of the PBS buffer is 7.2.

[0121] Step 3, Purification: The extract was centrifuged at 10000g for 10 minutes to remove impurities and precipitate. The supernatant was then filtered through a 100kDa ultrafiltration membrane, and the filtrate was collected. The filtrate was then centrifuged at 100000g for 70 minutes to obtain exosome precipitate. The exosome precipitate was purified exosomes containing a high concentration of saffron exosomes.

[0122] Step 4, Drying and Preservation: The purified exosomes are preserved by freeze-drying at -20℃ to ensure their stability and activity during long-term storage.

[0123] Example 9

[0124] A method for preparing saffron exosomes includes the following steps:

[0125] Step 1, Raw material processing: Collect high-quality saffron stigmas and petals, wash them with tap water, dry them at 0℃, and then pulverize them to 80 mesh to obtain saffron powder, in order to increase the efficiency of subsequent extraction.

[0126] High-quality saffron refers to saffron that is undamaged, disease-free, and normally developed.

[0127] Step 2, Extraction Process:

[0128] Saffron powder was soaked in a specific solvent at a material-to-liquid ratio of 1:6, and then subjected to ultrasonic extraction to obtain an extract. The ultrasonic power was set to 200W, the time to 30 minutes, and the temperature was controlled at 4℃.

[0129] The specific solvent formulation is: 0.015g potassium sulfate per liter of PBS buffer. The PBS buffer formulation per liter is as follows: 8g sodium chloride, 0.2g potassium chloride, 2.9g disodium hydrogen phosphate, 0.24g potassium dihydrogen phosphate, and deionized water to make up to 1L; the pH of the PBS buffer is 7.2.

[0130] Step 3, Purification: The extract was centrifuged at 10000g for 10 minutes to remove impurities and precipitate. The supernatant was then filtered through a 100kDa ultrafiltration membrane, and the filtrate was collected. The filtrate was then centrifuged at 100000g for 70 minutes to obtain exosome precipitate. The exosome precipitate was purified exosomes containing a high concentration of saffron exosomes.

[0131] Step 4, Drying and Preservation: The purified exosomes are preserved by freeze-drying at -20℃ to ensure their stability and activity during long-term storage.

[0132] Example 10

[0133] A method for preparing saffron exosomes includes the following steps:

[0134] Step 1, Raw material processing: Collect high-quality saffron stigmas and petals, wash them with tap water, dry them at 0℃, and then pulverize them to 80 mesh to obtain saffron powder, in order to increase the efficiency of subsequent extraction.

[0135] High-quality saffron refers to saffron that is undamaged, disease-free, and normally developed.

[0136] Step 2, Extraction Process:

[0137] Saffron powder was soaked in a specific solvent at a material-to-liquid ratio of 1:6, and then subjected to ultrasonic extraction to obtain an extract. The ultrasonic power was set to 200W, the time to 30 minutes, and the temperature was controlled at 4℃.

[0138] The specific solvent formulation is: 0.005 g potassium nitrate per liter of PBS buffer. The PBS buffer formulation per liter is as follows: 8 g sodium chloride, 0.2 g potassium chloride, 2.9 g disodium hydrogen phosphate, 0.24 g potassium dihydrogen phosphate, and deionized water to make up to 1 L; the pH of the PBS buffer is 7.2.

[0139] Add papain at a concentration of 0.5% of the extract mass to the extract and hydrolyze at 50°C for 2 hours to obtain the hydrolysate.

[0140] Step 3, Purification: The enzymatic hydrolysis product was centrifuged at 10000g for 10 minutes to remove impurities and precipitate. The supernatant was then filtered through a 100kDa ultrafiltration membrane, and the filtrate was collected. The filtrate was then centrifuged at 100000g for 70 minutes to obtain exosome precipitate. The exosome precipitate was purified exosomes containing a high concentration of saffron exosomes.

[0141] Step 4, Drying and Preservation: The purified exosomes are preserved by freeze-drying at -20℃ to ensure their stability and activity during long-term storage.

[0142] Example 11

[0143] A method for preparing saffron exosomes includes the following steps:

[0144] Step 1, Raw material processing: Collect high-quality saffron stigmas and petals, wash them with tap water, dry them at 0℃, and then pulverize them to 80 mesh to obtain saffron powder, in order to increase the efficiency of subsequent extraction.

[0145] High-quality saffron refers to saffron that is undamaged, disease-free, and normally developed.

[0146] Step 2, Extraction Process:

[0147] Saffron powder was soaked in a specific solvent at a material-to-liquid ratio of 1:6, and then subjected to ultrasonic extraction to obtain an extract. The ultrasonic power was set to 200W, the time to 30 minutes, and the temperature was controlled at 4℃.

[0148] The specific solvent formulation is: 0.005 g potassium nitrate per liter of PBS buffer. The PBS buffer formulation per liter is as follows: 8 g sodium chloride, 0.2 g potassium chloride, 2.9 g disodium hydrogen phosphate, 0.24 g potassium dihydrogen phosphate, and deionized water to make up to 1 L; the pH of the PBS buffer is 7.2.

[0149] Add 1% trypsin (by mass of extract) to each liter of extract and hydrolyze at 40°C for 1 hour to obtain the hydrolysate.

[0150] Step 3, Purification: The enzymatic hydrolysis product was centrifuged at 10000g for 10 minutes to remove impurities and precipitate. The supernatant was then filtered through a 100kDa ultrafiltration membrane, and the filtrate was collected. The filtrate was then centrifuged at 100000g for 70 minutes to obtain exosome precipitate. The exosome precipitate was purified exosomes containing a high concentration of saffron exosomes.

[0151] Step 4, Drying and Preservation: The purified exosomes are preserved by freeze-drying at -20℃ to ensure their stability and activity during long-term storage.

[0152] Compare with Example 1

[0153] A method for preparing saffron exosomes includes the following steps:

[0154] Step 1, Raw material processing: Collect high-quality saffron stigmas and petals, wash them with tap water, dry them at 0℃, and then pulverize them to 80 mesh to obtain saffron powder, in order to increase the efficiency of subsequent extraction.

[0155] High-quality saffron refers to saffron that is undamaged, disease-free, and normally developed.

[0156] Step 2, Extraction Process:

[0157] Saffron powder was soaked in a specific solvent at a material-to-liquid ratio of 1:6, and then subjected to ultrasonic extraction to obtain an extract. The ultrasonic power was set to 100W, the time to 30 minutes, and the temperature was controlled at 4℃.

[0158] The specific solvent formulation is: 0.005 g potassium nitrate per liter of PBS buffer. The PBS buffer formulation per liter is as follows: 8 g sodium chloride, 0.2 g potassium chloride, 2.9 g disodium hydrogen phosphate, 0.24 g potassium dihydrogen phosphate, and deionized water to make up to 1 L; the pH of the PBS buffer is 7.2.

[0159] Step 3, Purification: The extract was centrifuged at 10000g for 10 minutes to remove impurities and precipitate. The supernatant was then filtered through a 100kDa ultrafiltration membrane, and the filtrate was collected. The filtrate was then centrifuged at 100000g for 70 minutes to obtain exosome precipitate. The exosome precipitate was purified exosomes containing a high concentration of saffron exosomes.

[0160] Step 4, Drying and Preservation: The purified exosomes are preserved by freeze-drying at -20℃ to ensure their stability and activity during long-term storage.

[0161] Compare with Example 2

[0162] A method for preparing saffron exosomes includes the following steps:

[0163] Step 1, Raw material processing: Collect high-quality saffron stigmas and petals, wash them with tap water, dry them at 0℃, and then pulverize them to 80 mesh to obtain saffron powder, in order to increase the efficiency of subsequent extraction.

[0164] High-quality saffron refers to saffron that is undamaged, disease-free, and normally developed.

[0165] Step 2, Extraction Process:

[0166] Saffron powder was soaked in a specific solvent at a material-to-liquid ratio of 1:6, and then subjected to ultrasonic extraction to obtain an extract. The ultrasonic power was set to 200W, the time to 15 minutes, and the temperature was controlled at 4℃.

[0167] The specific solvent formulation is: 0.005 g potassium nitrate per liter of PBS buffer. The PBS buffer formulation per liter is as follows: 8 g sodium chloride, 0.2 g potassium chloride, 2.9 g disodium hydrogen phosphate, 0.24 g potassium dihydrogen phosphate, and deionized water to make up to 1 L; the pH of the PBS buffer is 7.2.

[0168] Step 3, Purification: The extract was centrifuged at 10000g for 10 minutes to remove impurities and precipitate. The supernatant was then filtered through a 100kDa ultrafiltration membrane, and the filtrate was collected. The filtrate was then centrifuged at 100000g for 70 minutes to obtain exosome precipitate. The exosome precipitate was purified exosomes containing a high concentration of saffron exosomes.

[0169] Step 4, Drying and Preservation: The purified exosomes are preserved by freeze-drying at -20℃ to ensure their stability and activity during long-term storage.

[0170] Compare with Example 3

[0171] A method for preparing saffron exosomes includes the following steps:

[0172] Step 1, Raw material processing: Collect high-quality saffron stigmas and petals, wash them with tap water, dry them at 0℃, and then pulverize them to 80 mesh to obtain saffron powder, in order to increase the efficiency of subsequent extraction.

[0173] High-quality saffron refers to saffron that is undamaged, disease-free, and normally developed.

[0174] Step 2, Extraction Process:

[0175] Saffron powder was soaked in a specific solvent at a material-to-liquid ratio of 1:6, and then subjected to ultrasonic extraction to obtain an extract. The ultrasonic power was set to 400W, the time to 30 minutes, and the temperature was controlled at 4℃.

[0176] The specific solvent formulation is: 0.005 g potassium nitrate per liter of PBS buffer. The PBS buffer formulation per liter is as follows: 8 g sodium chloride, 0.2 g potassium chloride, 2.9 g disodium hydrogen phosphate, 0.24 g potassium dihydrogen phosphate, and deionized water to make up to 1 L; the pH of the PBS buffer is 7.2.

[0177] Step 3, Purification: The extract was centrifuged at 10000g for 10 minutes to remove impurities and precipitate. The supernatant was then filtered through a 100kDa ultrafiltration membrane, and the filtrate was collected. The filtrate was then centrifuged at 100000g for 70 minutes to obtain exosome precipitate. The exosome precipitate was purified exosomes containing a high concentration of saffron exosomes.

[0178] Step 4, Drying and Preservation: The purified exosomes are preserved by freeze-drying at -20℃ to ensure their stability and activity during long-term storage.

[0179] Compare with Example 4

[0180] A method for preparing saffron exosomes includes the following steps:

[0181] Step 1, Raw material processing: Collect high-quality saffron stigmas and petals, wash them with tap water, dry them at 0℃, and then pulverize them to 80 mesh to obtain saffron powder, in order to increase the efficiency of subsequent extraction.

[0182] High-quality saffron refers to saffron that is undamaged, disease-free, and normally developed.

[0183] Step 2, Extraction Process:

[0184] Saffron powder was soaked in a specific solvent at a material-to-liquid ratio of 1:6, and then subjected to ultrasonic extraction to obtain the extract. The ultrasonic power was set to 200W, the time to 120 minutes, and the temperature was controlled at 4℃.

[0185] The specific solvent formulation is: 0.005 g potassium nitrate per liter of PBS buffer. The PBS buffer formulation per liter is as follows: 8 g sodium chloride, 0.2 g potassium chloride, 2.9 g disodium hydrogen phosphate, 0.24 g potassium dihydrogen phosphate, and deionized water to make up to 1 L; the pH of the PBS buffer is 7.2.

[0186] Step 3, Purification: The extract was centrifuged at 10000g for 10 minutes to remove impurities and precipitate. The supernatant was then filtered through a 100kDa ultrafiltration membrane, and the filtrate was collected. The filtrate was then centrifuged at 100000g for 70 minutes to obtain exosome precipitate. The exosome precipitate was purified exosomes containing a high concentration of saffron exosomes.

[0187] Step 4, Drying and Preservation: The purified exosomes are preserved by freeze-drying at -20℃ to ensure their stability and activity during long-term storage.

[0188] Compare with Example 5

[0189] A method for preparing saffron exosomes includes the following steps:

[0190] Step 1, Raw material processing: Collect high-quality saffron stigmas and petals, wash them with tap water, dry them at 0℃, and then pulverize them to 80 mesh to obtain saffron powder, in order to increase the efficiency of subsequent extraction.

[0191] High-quality saffron refers to saffron that is undamaged, disease-free, and normally developed.

[0192] Step 2, Extraction Process:

[0193] Saffron powder was soaked in a specific solvent at a material-to-liquid ratio of 1:6, and then subjected to ultrasonic extraction to obtain an extract. The ultrasonic power was set to 200W, the time to 30 minutes, and the temperature was controlled at 30℃.

[0194] The specific solvent formulation is: 0.005 g potassium nitrate per liter of PBS buffer. The PBS buffer formulation per liter is as follows: 8 g sodium chloride, 0.2 g potassium chloride, 2.9 g disodium hydrogen phosphate, 0.24 g potassium dihydrogen phosphate, and deionized water to make up to 1 L; the pH of the PBS buffer is 7.2.

[0195] Step 3, Purification: The extract was centrifuged at 10000g for 10 minutes to remove impurities and precipitate. The supernatant was then filtered through a 100kDa ultrafiltration membrane, and the filtrate was collected. The filtrate was then centrifuged at 100000g for 70 minutes to obtain exosome precipitate. The exosome precipitate was purified exosomes containing a high concentration of saffron exosomes.

[0196] Step 4, Drying and Preservation: The purified exosomes are preserved by freeze-drying at -20℃ to ensure their stability and activity during long-term storage.

[0197] Compare with Example 6

[0198] A method for preparing saffron exosomes includes the following steps:

[0199] Step 1, Raw material processing: Collect high-quality saffron stigmas and petals, wash them with tap water, dry them at 0℃, and then pulverize them to 80 mesh to obtain saffron powder, in order to increase the efficiency of subsequent extraction.

[0200] High-quality saffron refers to saffron that is undamaged, disease-free, and normally developed.

[0201] Step 2, Extraction Process:

[0202] Saffron powder was soaked in a solvent at a material-to-liquid ratio of 1:6, and then subjected to ultrasonic extraction to obtain an extract. The ultrasonic power was set to 200W, the time to 30 minutes, and the temperature was controlled at 4℃.

[0203] The solvent formulation is PBS buffer. The PBS buffer formulation per liter is as follows: 8g sodium chloride, 0.2g potassium chloride, 2.9g disodium hydrogen phosphate, 0.24g potassium dihydrogen phosphate, and deionized water to make up to 1L; the pH of the PBS buffer is 7.2.

[0204] Step 3, Purification: The extract was centrifuged at 10000g for 10 minutes to remove impurities and precipitate. The supernatant was then filtered through a 100kDa ultrafiltration membrane, and the filtrate was collected. The filtrate was then centrifuged at 100000g for 70 minutes to obtain exosome precipitate. The exosome precipitate was purified exosomes containing a high concentration of saffron exosomes.

[0205] Step 4, Drying and Preservation: The purified exosomes are preserved by freeze-drying at -20℃ to ensure their stability and activity during long-term storage.

[0206] Compare with Example 7

[0207] A method for preparing saffron exosomes includes the following steps:

[0208] Step 1, Raw material processing: Collect high-quality saffron stigmas and petals, wash them with tap water, dry them at 0℃, and then pulverize them to 80 mesh to obtain saffron powder, in order to increase the efficiency of subsequent extraction.

[0209] High-quality saffron refers to saffron that is undamaged, disease-free, and normally developed.

[0210] Step 2, Extraction Process:

[0211] Saffron powder was soaked in a solvent at a material-to-liquid ratio of 1:6, and then subjected to ultrasonic extraction to obtain an extract. The ultrasonic power was set to 100W, the time to 30 minutes, and the temperature was controlled at 4℃.

[0212] The solvent formulation is: deionized water.

[0213] Step 3, Purification: The extract was centrifuged at 10000g for 10 minutes to remove impurities and precipitate. The supernatant was then filtered through a 100kDa ultrafiltration membrane, and the filtrate was collected. The filtrate was then centrifuged at 100000g for 70 minutes to obtain exosome precipitate. The exosome precipitate was purified exosomes containing a high concentration of saffron exosomes.

[0214] Step 4, Drying and Preservation: The purified exosomes are preserved by freeze-drying at -20℃ to ensure their stability and activity during long-term storage.

[0215] The purity of exosomes in exosome solutions (after resuspending the exosome precipitate to 100 μg / mL) was determined using a nanoparticle size analyzer. The method for calculating exosome purity was as follows: first, the total number of vesicles (A) in the exosome solution was measured; then, the number of vesicles with a size between 30 nm and 150 nm (B) was calculated. The exosome purity was calculated as the ratio of B to A multiplied by 100%. Three replicates were performed for each group, and the average value was calculated to determine the standard deviation.

[0216] The differences in parameters and particle size statistics between the various embodiments and the control examples are as follows:

[0217] The difference between Examples 1 to 4 lies in the different ultrasonic conditions, and the difference between Examples 5 to 9 lies in the different specific solvent formulations. The particle size range of the exosome precipitates extracted in Examples 1 to 9 is mainly concentrated in the range of 30 nm to 150 nm, with the particle size of 90 nm to 110 nm accounting for more than 50%.

[0218] In Examples 10 and 11, due to the addition of a biomodification step and enzymatic hydrolysis, the particle size of the exosome precipitate was reduced, mainly clustered in the range of 30 nm to 50 nm, with a small amount distributed in the range of 50 nm to 150 nm.

[0219] In Control Examples 1 and 2, due to the low ultrasonic power or short duration, the cell structure could not be effectively destroyed, and the exosomes could not be released, resulting in low exosome purity and correspondingly low extraction efficiency. Exosomes with particle sizes mainly ranging from 30 nm to 100 nm were extracted, and the number of exosomes between 100 nm and 150 nm was even smaller.

[0220] In contrast, in Control Examples 3 to 5, the excessively high temperature, prolonged duration, or high temperature of the ultrasound not only damaged the cell structure but also the exosome structure, and may even have led to exosome structural remodeling. The particle size range of the extracted exosome precipitates was mainly concentrated in the range of 30 nm to 150 nm, with particles in the range of 80 nm to 110 nm accounting for more than 50%, and the antibacterial and antioxidant biological activities were significantly reduced.

[0221] Comparative Examples 6 and 7 are results of other solvent formulations. By comparing the exosome purity of Comparative Examples 6 and 7 with that of each example, it can be seen that when PBS and potassium sulfate or potassium nitrate are present in the reagent, the number of exosomes stimulated to be secreted is the highest, and the purity of the obtained exosomes is also the highest.

[0222] Specifically, the exosome purity and particle size distribution results of different embodiments and control examples are shown in Table 1.

[0223] Table 1. Results of exosome purity and particle size distribution

[0224]

[0225] In vitro antibacterial test:

[0226] The antibacterial rate was statistically analyzed using existing techniques. Test strains of various bacteria were prepared, including *Escherichia coli* ATCC25922, *Staphylococcus aureus* ATCC27733, and drug-resistant *Staphylococcus aureus* VISA. After one activation, a concentration of 10⁻⁶ was prepared. 8 For a bacterial suspension with a concentration of [number] cells / mL, 0.1 g of saffron exosomes were added to every 100 mL of bacterial suspension and allowed to incubate for 30 min. The viable bacterial concentration before and after incubation was determined using a live bacteria technique, and the inhibition rate was calculated. Three parallel experiments were performed, and the average value was taken. An equal volume of sterile water was used as a blank control instead of saffron exosomes.

[0227] Antibacterial rate (%) = 100 * [(Viable bacterial concentration before treatment - Viable bacterial concentration after treatment) / Bacterial concentration before treatment].

[0228] In vitro free radical scavenging assay (DPPH method):

[0229] Weigh DPPH in the dark and prepare a 0.1 mmol / L DPPH working solution with anhydrous ethanol. Prepare and use immediately, and store in the dark.

[0230] Add the relevant reagents to each well of the 96-well plate as follows:

[0231] A0: Absorbance of 100 μL anhydrous ethanol + 100 μL DPPH working solution, i.e. blank control;

[0232] A1: Absorbance of 100 μL of test sample + 100 μL of DPPH working solution;

[0233] Set up 3 duplicate wells for each group, and gently tap to mix. Let stand at room temperature in the dark for 30 min. Measure the absorbance at 517 nm and record A0 and A1. Calculate the clearance rate.

[0234] Clearance rate (%) = [(A0 - A1) / A0] × 100.

[0235] Table 2. Statistical results of bacterial inhibition rate

[0236]

[0237] The results in Table 2 show that the exosomes prepared by the method in the control example have significantly reduced activity, such as antibacterial effect and antioxidant effect of scavenging free radicals. This indicates that the preparation process has a significant impact on the activity of exosomes.

[0238] Biomodification can be achieved by treating exosomes with bioenzymes to change the structure or composition of exosome surface proteins, reduce exosome particle size, and endow them with new biological activities, such as enhanced antibacterial effects (see data from Examples 10 and 11 in Table 2). It still exhibits antibacterial effects against some drug-resistant bacteria.

[0239] It should be noted that although the present invention conducted in vitro antibacterial experiments and in vitro free radical scavenging experiments, the results of these two experiments show that the saffron exosomes prepared by the present invention have the potential for antibacterial application and free radical scavenging, and may also have relevant application value in vivo.

[0240] It should be noted that when numerical ranges are involved in this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described in this invention to avoid redundancy. Although preferred embodiments of this invention have been described, those skilled in the art, once they understand the inventive concept of this invention, can make other changes and modifications to these embodiments, and all such changes and modifications fall within the scope of this invention.

[0241] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. If such modifications and variations fall within the scope of equivalents of this invention, then this invention also intends to include these modifications and variations.

Claims

1. A method for preparing saffron exosomes, characterized in that, Includes the following steps: Collect stigmas and petals of saffron, wash and dry them, then grind them to 80-100 mesh to obtain saffron powder; Saffron powder was soaked in a specific solvent at a material-to-liquid ratio of 1:6 to 10, and then subjected to ultrasonic extraction to obtain an extract. The ultrasonic power was set to 200W to 300W, the time to 30 to 60 minutes, and the temperature was controlled at 4℃ to 8℃. The specific solvent formula was as follows: 0.005g to 0.01g of potassium nitrate or 0.01g to 0.02g of potassium sulfate per liter of PBS buffer. The extract was centrifuged at 8000g~10000g to remove impurities and precipitates. The supernatant was filtered through a 100kDa~120kDa ultrafiltration membrane, and the filtrate was collected. The filtrate was then centrifuged at 100000g~110000g for 70 minutes~90 minutes to obtain exosome precipitates, which are saffron exosomes.

2. The method for preparing saffron exosomes according to claim 1, characterized in that, The mass of potassium nitrate added per liter of PBS buffer is 0.007 g. The mass of potassium sulfate added per liter of PBS buffer is 0.015 g.

3. The method for preparing saffron exosomes according to claim 1, characterized in that, The PBS buffer formulation per liter is as follows: 8g sodium chloride, 0.2g potassium chloride, 2.9g disodium hydrogen phosphate, 0.24g potassium dihydrogen phosphate, and deionized water to make up to 1L; The pH of the PBS buffer is 7.2-7.

4.

4. The method for preparing saffron exosomes according to claim 1, characterized in that, Exosome precipitate was freeze-dried to obtain exosome lyophilized powder.

5. The method for preparing saffron exosomes according to claim 1, characterized in that, It also includes a biomodification step of exosomes, the method of which is as follows: add 0.5% to 1% of protease by mass of the extract to the extract, and enzymatically hydrolyze at 40℃ to 50℃ for 1 to 2 hours to obtain the enzymatic hydrolysis product; After obtaining the enzymatic hydrolysis product, a centrifugation process at a speed of 8000g~10000g is performed to remove impurities and precipitates.

6. The method for preparing saffron exosomes according to claim 5, characterized in that, The protease is either trypsin or papain.

7. The method for preparing saffron exosomes according to claim 1, characterized in that, The conditions for cleaning and drying are: washing with tap water and drying at 0℃.

8. The application of saffron exosomes prepared by the preparation method according to any one of claims 1 to 7 in the preparation of antibacterial agents, characterized in that, The antibacterial agent is effective against Escherichia coli ( Escherichia coli Staphylococcus aureus ( staphylococcus aureus ) and drug-resistant Staphylococcus aureus VISA.

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