Radix astragali exosome with anti-breast cancer and anti-lung cancer efficacy, preparation method and application thereof

Abstract

This invention provides Astragalus exosomes with anti-breast cancer and anti-lung cancer effects, their preparation method, and applications, relating to the field of biomedical technology. The preparation method of the Astragalus exosomes includes the following steps: Astragalus root is soaked in physiological saline to obtain an Astragalus suspension; homogenization and centrifugation are performed to obtain supernatant 1, which is then filtered through a filter membrane to obtain supernatant 2; tangential flow filtration and concentration are performed: supernatant 2 is continuously filtered and concentrated through a 0.15-0.3 μm tangential flow filtration membrane assembly, the permeate is collected, and the retained material is discarded; the permeate is then continuously filtered and concentrated through a 0.015-0.025 μm tangential flow filtration membrane assembly, the retained liquid is collected, and the permeate is discarded; the retained liquid is filtered through a 100-300 kDa ultrafiltration membrane to retain the exosomes, and the concentration is performed to obtain a concentrated solution; washing, filtration, purification, and sterilization are then performed. The Astragalus exosomes prepared by this invention have significant inhibitory effects on both breast cancer and lung cancer, solving the problems of unclear anti-cancer active ingredients and low utilization rate of traditional Astragalus extracts.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to Astragalus exosomes with anti-breast cancer and anti-lung cancer effects, their preparation methods, and applications. Background Technology

[0002] Breast cancer and lung cancer are among the most common malignant tumors worldwide. Breast cancer ranks first and second in incidence among women in my country, while lung cancer has the highest mortality rate. Both face challenges such as strong side effects of treatment drugs, significant drug resistance, and a lack of natural active drugs. Plant exosomes, commonly known as plant-derivedexosome-like nanoparticles (PELNs), are a class of 30-300 nm membrane vesicles isolated from plant tissues, containing proteins, lipids, and nucleic acids. As components of plants, PELNs play a vital role in plant metabolism, physiological function maintenance, and disease defense. They can also regulate cellular activity across species and mediate communication between different species. They have significant applications and functions in physiological processes, disease prevention and treatment, and nutrient supply, enabling disease intervention and drug delivery. Compared to animal exosomes, PELNs have the advantages of widely available raw materials and ease of large-scale production, showing broad application prospects in the biomedical field.

[0003] Publicly available patents related to plant exosomes, such as Chinese patent application CN120230697A, disclose a wolfberry exosome and its preparation method and application. The method includes the following steps: (a) obtaining plant material; (b) pretreating the obtained plant material to obtain a crude plant extract; and (c) performing layer-by-layer filtration on the obtained crude plant extract. In step (c), the layer-by-layer filtration includes a deep filtration step and an optional mulch film filtration step. Chinese patent application CN120485092A discloses a ginseng exosome, its extraction method, and its application in the preparation of anti-photoaging skin care products. The extraction method includes the following steps: S1: Using distilled water to remove the surface dirt of ginseng until the surface is clean and free of dirt, the ginseng is cut into small pieces and juiced using a juicer to obtain crude ginseng juice; the ginseng is five-year-old cultivated ginseng or fifteen-year-old or older wild ginseng; S2: The crude ginseng juice obtained in S1 is centrifuged in a centrifuge pre-cooled to 4°C to obtain ginseng stock solution; S3: Chitosan solution is added to the ginseng stock solution obtained in S2, heated and stirred, allowed to stand at room temperature for 2 hours, centrifuged at 10000×g for 10-30 minutes, the precipitate is discarded, and a clear ginseng liquid is obtained; S4: A tangential flow ultrafiltration system is used, and a hollow fiber column is selected to ultrafilter and concentrate the ginseng liquid obtained in S3 to obtain ginseng exosomes. None of the aforementioned existing technologies involve Astragalus exosomes, nor have they conducted specific anti-cancer verification for breast cancer and lung cancer, and they lack quantitative activity data based on zebrafish tumor transplantation models.

[0004] Astragalus, a traditional Chinese medicine, has been shown to possess anti-tumor potential in its polysaccharides and glycosides. However, current research largely focuses on crude extracts, resulting in unclear active ingredients, ambiguous mechanisms of action, and low bioavailability, which hinders the development of precision anti-cancer drugs. Furthermore, research on astragalus exosomes is extremely limited, and there is currently no standardized preparation process for astragalus exosomes with proven anti-cancer efficacy validated in animal models.

[0005] Furthermore, existing astragalus extract preparation processes mostly employ traditional methods such as water extraction and alcohol precipitation, which cannot effectively separate and purify exosomes, leading to loss of active ingredients or interference from impurities. Simultaneously, the lack of quality control standards for astragalus exosomes makes it difficult to guarantee product stability and consistency in anticancer activity. Therefore, developing a stable, quality-controllable astragalus exosome formulation that has been validated for efficacy against breast and lung cancer using a zebrafish model is crucial to overcoming current technological bottlenecks. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides an Astragalus exosome and its preparation method. The method combines raw material pretreatment, high-pressure homogenization and cell wall disruption, purification, tangential flow filtration concentration, ultrafiltration concentration, washing, and sterilization. The resulting Astragalus exosome exhibits significant inhibitory effects against both breast and lung cancer. This solves the problems of unclear anticancer active ingredients and low utilization rate in traditional Astragalus extracts. The provided preparation process stably preserves the biological activity of exosomes, ensuring controllable product quality and providing a new material basis for the development of anti-breast and anti-lung cancer drugs.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] On one hand, the present invention provides a method for preparing Astragalus exosomes, comprising the following steps:

[0009] S1: Pretreatment: Soak Astragalus root in physiological saline to obtain Astragalus suspension;

[0010] S2: Cell wall disruption extraction: Astragalus suspension is homogenized 1-3 times under 80-100MPa pressure to obtain homogenized suspension;

[0011] S3: Preliminary purification: Centrifuge the homogenized suspension to obtain supernatant 1, then filter it through a filter membrane to obtain supernatant 2;

[0012] S4: Tangential flow filtration concentration:

[0013] S4-1: The supernatant 2 is continuously filtered and concentrated at 3-5℃ through a 0.15-0.3μm tangential flow filtration membrane module. The permeate is collected and the retained material is discarded.

[0014] S4-2: The permeate is then continuously filtered and concentrated at 3-5℃ through a 0.015-0.025μm tangential flow filtration membrane module. The retentate is collected and the permeate is discarded.

[0015] S5: Ultrafiltration Concentration: The retentate is filtered through a 100-300kDa ultrafiltration membrane to retain exosomes and then concentrated to obtain a concentrated solution;

[0016] S6: Washing and filtration: Use PBS buffer as the washing solution, wash and filtration the concentrated solution 2-5 times, concentrate, and obtain the pure concentrated solution of Astragalus exosomes;

[0017] S7: Purification and sterilization: Aseptic filtration of the pure extract of Astragalus exosomes yields the product.

[0018] Preferably, in step S1, the Astragalus root is the root of an Astragalus plant belonging to the genus Astragalus of the legume family that is free from mold and pests.

[0019] Preferably, in step S1, the Astragalus root needs to be washed, dried, crushed, and sieved.

[0020] More preferably, in step S1, the Astragalus root is rinsed with purified water and then dried, the raw material moisture content is ≤12%, and it is crushed and passed through a 40-60 mesh sieve.

[0021] Preferably, in step S1, the mass-to-volume ratio of Astragalus root to physiological saline is 1g:5-10mL.

[0022] Preferably, in step S1, the soaking is performed by soaking at 3-5℃ for 12-24 hours, stirring for 10-15 minutes every 6 hours during the soaking period, to obtain an Astragalus suspension.

[0023] Preferably, in step S2, the homogenization pressure is 80-100 MPa, the number of times is 1-3, and the temperature is <10℃.

[0024] Preferably, in step S3, the relative centrifugal force is 500-10000×g, the temperature is 3-5℃, the time is 15-45min, and the number of times is 1-3.

[0025] More preferably, in step S3, the centrifugation includes the following steps: the homogenized suspension is centrifuged for the first time at 500×g and 4℃ for 15min, and the precipitate (tissue residue) is discarded; the suspension is centrifuged for the second time at 1000×g and 4℃ for 45min, and the precipitate (organelles) is discarded, to obtain supernatant 1; after each centrifugation, the supernatant is carefully transferred to avoid disturbing the precipitate.

[0026] Preferably, in step S3, the pore size of the filter membrane is 0.22-0.45 μm;

[0027] Preferably, in step S3, the filtering is performed 1-3 times.

[0028] More preferably, in step S3, the filtration includes the following steps: supernatant 1 is passed through a 0.45μm filter membrane to remove macromolecular impurities, resulting in filtrate; the filtrate is then passed through a 0.22μm filter membrane to obtain supernatant 2.

[0029] S4-1: The supernatant 2 is continuously filtered and concentrated at 3-5℃ through a 0.15-0.3μm tangential flow filtration membrane module. The permeate is collected and the retained material is discarded.

[0030] S4-2: The permeate is then continuously filtered and concentrated at 3-5℃ through a 0.015-0.025μm tangential flow filtration membrane module. The retentate is collected and the permeate is discarded.

[0031] Preferably, in steps S4-1 and S4-2, the transmembrane pressure (TMP) of the continuous filtration is 0.05-0.2MPa, and the tangential flow velocity (CFV) is 1.0-1.5m / s.

[0032] Preferably, in step S5, the concentration is to concentrate to 1 / 10-1 / 20 of the original volume.

[0033] Preferably, in step S6, the washing and filtration includes the following steps: maintaining the transmembrane pressure (TMP) at 0.1-0.15 MPa and the tangential flow rate (CFV) at 1.2-1.8 m / s, adding sterile PBS buffer (pH 7.2-7.4) to the concentrate, the volume of the sterile PBS buffer being 5-8 times that of the concentrate, adding it in 3-4 portions, and circulating and filtering for 10-15 min after each addition.

[0034] Preferably, in step S7, the aseptic filtration includes the following steps: the concentrated extract of Astragalus exosomes is passed sequentially through filter membranes of 0.4-0.5μm and 0.2-0.3μm, and the filtrate is collected to obtain Astragalus exosomes.

[0035] Preferably, the particle size of the Astragalus exosomes is 30-150 nm.

[0036] Preferably, the preparation method may further include the following steps:

[0037] Astragalus exosomes are mixed with a lyophilization protectant solution, equilibrated, and then lyophilized to obtain lyophilized powder of astragalus exosomes.

[0038] More preferably, it includes the following steps:

[0039] Slowly mix the sterile solution with an equal volume of lyophilization protectant solution of twice the concentration. The protectant is 3%-5% (w / v) trehalose. Equilibrate at 3-5°C for 20-40 minutes. Aliquot the mixed sample solution into lyophilization vials and rapidly freeze them in a -80°C ultra-low temperature freezer or the cold trap of a freeze dryer. Lyophilization parameters: condenser temperature: ≤-50°C, vacuum degree: 10-50 Pa (0.1-0.5 mbar), duration: approximately 20-40 hours (depending on the volume of liquid and the container).

[0040] When storing freeze-dried products, high-purity nitrogen should be introduced and the product sealed with a stopper to isolate it from oxygen and moisture. Storage conditions: Freeze-dried powder should be stored in a light-proof, dry environment at ≤-20℃.

[0041] Secondly, the present invention provides Astragalus exosomes prepared by the aforementioned preparation method.

[0042] Preferably, the Astragalus exosomes meet the following quality standards: protein impurity content ≤ 5 wt%, polysaccharide impurity content ≤ 3 wt%, and endotoxin content ≤ 0.5 EU / mL.

[0043] Thirdly, the present invention provides the application of the aforementioned Astragalus exosomes in the preparation of drugs for treating lung cancer.

[0044] Preferably, the active component of the drug includes Astragalus exosomes prepared by the above preparation method.

[0045] More preferably, the mass percentage of Astragalus exosomes in the drug is 5%-20%.

[0046] Preferably, the dosage form of the drug is an injection, an oral preparation, or a topical preparation.

[0047] Preferably, the drug further includes a pharmaceutically acceptable carrier.

[0048] Furthermore, the pharmaceutically acceptable carriers include, but are not limited to, excipients, buffers, emulsifiers, stabilizers, diluents, binders, preservatives, lubricants, pH adjusters, cryoprotectants, flavoring agents, fillers, and antioxidants.

[0049] Preferably, the excipient is selected from at least one of microcrystalline cellulose, lactose, pregelatinized starch, cyclodextrin, carboxymethyl cellulose, mannitol, magnesium stearate, starch, calcium phosphate, ethyl cellulose, methyl cellulose, alginate, gelatin, gum arabic, glyceryl monostearate, sodium glycolate, guar gum, glycerol, and propylene glycol.

[0050] Preferably, the buffer is selected from at least one of sodium dihydrogen phosphate, sodium bicarbonate, ammonium bicarbonate, sodium acetate, citrate, histidine, and succinate.

[0051] Preferably, the emulsifier is selected from at least one of magnesium stearate, zinc stearate, calcium stearate, glyceryl stearate, sorbitan isostearate, sorbitan oleate, and polyglycerol-3 polyricinoleate.

[0052] Preferably, the stabilizer is selected from at least one of farnesian gum, agar, alginate, cellulose ether, and carboxymethyl chitosan.

[0053] Preferably, the diluent is selected from at least one of erythritol, mannitol, sorbitol, xylitol, lactose, sucrose, corn starch, potato starch, calcium phosphate, calcium citrate, and crystalline cellulose.

[0054] Preferably, the adhesive is selected from at least one of ethanol, starch paste, pregelatinized starch, dextrin, syrup, hydroxypropyl methylcellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyvinyl alcohol, polyethylene glycol, sodium alginate, polyvinylpyrrolidone, gum arabic, gelatin, and alginic acid.

[0055] Preferably, the preservative is selected from at least one of methylparaben, propylparaben, methylparaben, ethylparaben, propylparaben, chlorobutanol, phenoxyethanol, chlorhexidine, benzoic acid, sodium benzoate, chlorocresol, benzalkonium bromide, benzalkonium chloride, and ethylparaben.

[0056] Preferably, the lubricant is selected from at least one of magnesium stearate, zinc stearate, glyceryl monostearate, polyethylene glycol, stearic acid, talc, sodium chloride, sodium oleate, sodium lauryl sulfate, magnesium lauryl sulfate, sodium stearate fumarate, and poloxamer.

[0057] Preferably, the pH adjuster is selected from at least one of citric acid, fumaric acid, succinic acid, tartaric acid, malic acid, and ascorbic acid.

[0058] Preferably, the flavoring agent is selected from at least one of sweet orange flavoring, vanilla flavoring, strawberry flavoring, milk flavoring, banana flavoring, and cherry flavoring.

[0059] Preferably, the filler is selected from at least one of mannitol, xylitol, sorbitol, maltose, microcrystalline cellulose, glucose, lactose, sucrose, dextrin, starch, sodium alginate, and sodium bicarbonate.

[0060] Preferably, the antioxidant may be selected from at least one of L-cysteine ​​hydrochloride, L-cysteine ​​base, 4,4-(2,3-dimethyltetramethylenediamine), tocopherol-rich extracts (natural vitamin E), α-tocopherol (synthetic vitamin E), β-tocopherol, 6-tocopherol, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate, octyl gallate, dodecyl gallate, tert-butylhydroquinone (TBHQ), fumaric acid, malic acid, ascorbic acid (vitamin C), sodium ascorbate, calcium ascorbate, potassium ascorbate, ascorbate palmitate, and ascorbate stearate.

[0061] Fourthly, the present invention provides the application of the aforementioned Astragalus exosomes in the preparation of drugs for treating breast cancer.

[0062] Preferably, the active component of the drug includes Astragalus exosomes prepared by the above preparation method.

[0063] More preferably, the mass percentage of Astragalus exosomes in the drug is 5%-20%.

[0064] Preferably, the dosage form of the drug is an injection, an oral preparation, or a topical preparation.

[0065] Preferably, the drug further includes a pharmaceutically acceptable carrier.

[0066] Furthermore, the pharmaceutically acceptable carriers include, but are not limited to, excipients, buffers, emulsifiers, stabilizers, diluents, binders, preservatives, lubricants, pH adjusters, cryoprotectants, flavoring agents, fillers, and antioxidants.

[0067] Preferably, the excipient is selected from at least one of microcrystalline cellulose, lactose, pregelatinized starch, cyclodextrin, carboxymethyl cellulose, mannitol, magnesium stearate, starch, calcium phosphate, ethyl cellulose, methyl cellulose, alginate, gelatin, gum arabic, glyceryl monostearate, sodium glycolate, guar gum, glycerol, and propylene glycol.

[0068] Preferably, the buffer is selected from at least one of sodium dihydrogen phosphate, sodium bicarbonate, ammonium bicarbonate, sodium acetate, citrate, histidine, and succinate.

[0069] Preferably, the emulsifier is selected from at least one of magnesium stearate, zinc stearate, calcium stearate, glyceryl stearate, sorbitan isostearate, sorbitan oleate, and polyglycerol-3 polyricinoleate.

[0070] Preferably, the stabilizer is selected from at least one of farnesian gum, agar, alginate, cellulose ether, and carboxymethyl chitosan.

[0071] Preferably, the diluent is selected from at least one of erythritol, mannitol, sorbitol, xylitol, lactose, sucrose, corn starch, potato starch, calcium phosphate, calcium citrate, and crystalline cellulose.

[0072] Preferably, the adhesive is selected from at least one of ethanol, starch paste, pregelatinized starch, dextrin, syrup, hydroxypropyl methylcellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyvinyl alcohol, polyethylene glycol, sodium alginate, polyvinylpyrrolidone, gum arabic, gelatin, and alginic acid.

[0073] Preferably, the preservative is selected from at least one of methylparaben, propylparaben, methylparaben, ethylparaben, propylparaben, chlorobutanol, phenoxyethanol, chlorhexidine, benzoic acid, sodium benzoate, chlorocresol, benzalkonium bromide, benzalkonium chloride, and ethylparaben.

[0074] Preferably, the lubricant is selected from at least one of magnesium stearate, zinc stearate, glyceryl monostearate, polyethylene glycol, stearic acid, talc, sodium chloride, sodium oleate, sodium lauryl sulfate, magnesium lauryl sulfate, sodium stearate fumarate, and poloxamer.

[0075] Preferably, the pH adjuster is selected from at least one of citric acid, fumaric acid, succinic acid, tartaric acid, malic acid, and ascorbic acid.

[0076] Preferably, the flavoring agent is selected from at least one of sweet orange flavoring, vanilla flavoring, strawberry flavoring, milk flavoring, banana flavoring, and cherry flavoring.

[0077] Preferably, the filler is selected from at least one of mannitol, xylitol, sorbitol, maltose, microcrystalline cellulose, glucose, lactose, sucrose, dextrin, starch, sodium alginate, and sodium bicarbonate.

[0078] Preferably, the antioxidant may be selected from at least one of L-cysteine ​​hydrochloride, L-cysteine ​​base, 4,4-(2,3-dimethyltetramethylenediamine), tocopherol-rich extracts (natural vitamin E), α-tocopherol (synthetic vitamin E), β-tocopherol, 6-tocopherol, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate, octyl gallate, dodecyl gallate, tert-butylhydroquinone (TBHQ), fumaric acid, malic acid, ascorbic acid (vitamin C), sodium ascorbate, calcium ascorbate, potassium ascorbate, ascorbate palmitate, and ascorbate stearate.

[0079] Compared with the prior art, the present invention has the following beneficial effects:

[0080] (1) Clear and targeted activity: For the first time, based on a zebrafish model report (anti-breast cancer, anti-lung cancer), the inhibitory effect of Astragalus exosomes on two common tumors was quantitatively verified, and its regulatory effect on the inflammation-related gene il6 was clarified, making up for the deficiency of the existing astragalus extract's vague anti-cancer activity.

[0081] (2) Standardization of preparation process: The tangential flow filtration process is the ideal path for the standardized and large-scale production of Astragalus exosomes. It not only greatly improves production efficiency and product yield, but also reduces the technical dependence of operators, laying a solid technical foundation for the industrial production of Astragalus exosomes as functional food raw materials or drugs in the future.

[0082] (3) High quality controllability: Establish a complete quality standard covering physicochemical properties, purity, activity and stability. The activity index is directly related to the data of two zebrafish experimental reports to ensure the consistency of efficacy of each batch of products.

[0083] Outstanding innovation: Compared with existing plant exosome patents, this invention focuses on the anti-breast cancer and anti-lung cancer applications of Astragalus exosomes, and uses specific zebrafish model data as the core support. The process adds targeted stabilizers to solve the industry problems of aggregation and activity loss during exosome purification, making the application scenarios clearer. Attached Figure Description

[0084] Figure 1 This is an electron micrograph of the exosomes of Astragalus membranaceus in Example 1.

[0085] Figure 2 This is an electron micrograph of the exosomes of Astragalus membranaceus in Example 2.

[0086] Figure 3 This is a phenotypic diagram of zebrafish organs after treatment with Astragalus exosomes in Experiment Example 1.

[0087] Figure 4 The image shows the phenotype of renal edema in zebrafish induced by Astragalus exosomes in Experiment Example 1.

[0088] Figure 5 This is a typical fluorescence intensity diagram of zebrafish lung cancer cells after treatment with Astragalus exosomes in Experiment Example 1.

[0089] Figure 6 This is a pixel image showing the fluorescence intensity of lung cancer in zebrafish after treatment with Astragalus exosomes in Experiment Example 1. Detailed Implementation

[0090] To make the technical means, creative features, achieved objectives, and effects of this invention readily understandable, the invention is further illustrated below with specific embodiments. However, these embodiments are merely preferred embodiments and not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments described herein without creative effort are all within the scope of protection of this invention. It is worth noting that the raw materials used in this invention are all common commercially available products, and their sources are not specifically limited. The technical and scientific terms used in the embodiments have the meanings commonly understood by those skilled in the art to which this invention pertains.

[0091] Example 1

[0092] A method for preparing Astragalus exosomes includes the following steps:

[0093] S1. Raw material pretreatment: Select 500g of Astragalus root free from mold and pests, rinse with purified water and dry. The moisture content of the raw material is ≤12%. After crushing, pass through a 40-mesh sieve, add 4L of physiological saline with pH 7.2, and soak at 4℃ for 15h. Stir for 15min every 6h during the soaking period to obtain Astragalus suspension.

[0094] S2. Cell wall disruption extraction: The Astragalus suspension was placed in a high-pressure homogenizer and homogenized three times at 80 MPa pressure, with the temperature maintained below 10℃ to prevent degradation of bioactivity. After homogenization, it was allowed to stand at 4℃ for 2 hours.

[0095] S3. Preliminary purification: Centrifuge at 500×g for 15 min at 4℃, discard the precipitate (tissue residue), and centrifuge the supernatant again at 10000×g for 45 min at 4℃, discarding the precipitate (organelles). Carefully transfer the supernatant after each centrifugation step, avoiding disturbing the precipitate. Filter the final supernatant through a 0.45μm filter membrane to remove macromolecular impurities, obtaining the filtrate; then filter the filtrate through a 0.22μm filter membrane.

[0096] S4. Tangential flow filtration concentration: The filtered clear liquid is passed into a tangential flow filtration system equipped with a 0.15μm polyethersulfone (PES) hollow fiber membrane. The material is continuously filtered at 4℃, with the transmembrane pressure (TMP) controlled at 0.08MPa and the tangential flow rate (CFV) at 1.0m / s until the volume of the concentrated liquid is about 50mL. The permeate (containing exosomes and small molecules) is collected, and the retentate (subcellular debris and large protein aggregates) is discarded. The permeate is then continuously filtered and concentrated through a 0.02μm tangential flow filtration membrane module at 4℃. The retentate is collected, the permeate is discarded, and the 20-150nm Astragalus exosomes are enriched.

[0097] S5. Ultrafiltration Concentration: Using a 100kDa PES ultrafiltration membrane, exosomes are retained while small molecule impurities are allowed to pass through. The material is concentrated to 1 / 10 of its original volume at 4°C.

[0098] S6. Washing and Filtration: Maintaining stable TMP and CFV parameters from step S5, add sterile PBS buffer (pH 7.2), in a total volume 6 times the volume of the concentrate, in 4 portions. After each addition, circulate and filter for 15 minutes, then continue concentrating. Wash and filter until the conductivity of the permeate is consistent with that of PBS (≤20 μS / cm). The final concentrate volume is controlled at 8 mL to obtain a pure concentrate of Astragalus exosomes.

[0099] S7. Purification and sterilization: The concentrated extract of Astragalus exosomes was aseptically filtered through 0.45μm and 0.22μm PES membranes in sequence. The filtrate was collected and dispensed into sterile centrifuge tubes to obtain sterile solution.

[0100] S8. Freeze-drying preparation and preservation: Slowly mix the sterile solution with an equal volume of 2 times the concentration of the freeze-drying protectant solution. The protectant used is 4% (w / v) trehalose. Equilibrate at 4°C for 30 minutes. Aliquot the mixed sample solution into freeze-drying bottles and place them in a -80°C ultra-low temperature freezer or the cold trap of a freeze dryer for rapid freezing. Freeze-drying parameters: condenser temperature: ≤ -50°C, vacuum degree: 50 Pa (0.5 mbar), duration: approximately 30 hours, yielding 8.6 g of Astragalus exosome powder.

[0101] When storing freeze-dried products, high-purity nitrogen should be introduced and the product sealed with a stopper to isolate it from oxygen and moisture. Storage conditions: Freeze-dried powder should be stored in a light-proof, dry environment at ≤ -20℃.

[0102] Example 2

[0103] A method for preparing Astragalus exosomes includes the following steps:

[0104] S1. Raw material pretreatment: Select 500g of Astragalus root free from mold and pests, rinse with purified water and dry. The moisture content of the raw material is ≤12%. After crushing, pass through a 60-mesh sieve, add 2.5L of physiological saline with pH 7.4, and soak at 3℃ for 24h. Stir for 10min every 6h during the soaking period to obtain Astragalus suspension.

[0105] S2. Cell-wall breaking extraction: The Astragalus suspension was placed in a high-pressure homogenizer and homogenized twice at 80 MPa pressure, with the temperature maintained below 10℃ to prevent degradation of bioactivity. After homogenization, it was allowed to stand at 5℃ for 2 hours to obtain the homogenized suspension.

[0106] S3. Preliminary Purification: The homogenized suspension was centrifuged at 500×g for 15 min at 5℃, and the precipitate (tissue residue) was discarded. The supernatant was then centrifuged at 10000×g for 45 min at 3℃, and the precipitate (organelles) was discarded. After each centrifugation step, the supernatant was carefully transferred to avoid disturbing the precipitate. The final supernatant was filtered through a 0.45μm filter membrane to remove macromolecular impurities, yielding the filtrate; the filtrate was then filtered through a 0.22μm filter membrane.

[0107] S4. Tangential flow filtration concentration: The filtered clear liquid is passed into a tangential flow filtration system equipped with a 0.2μm polyethersulfone (PES) hollow fiber membrane. The material is continuously filtered at 3℃, with the transmembrane pressure (TMP) controlled at 0.2MPa and the tangential flow rate (CFV) at 1.5m / s until the volume of the concentrated liquid is about 50mL. The permeate (containing exosomes and small molecules) is collected, and the retentate (subcellular debris and large protein aggregates) is discarded. The permeate is then continuously filtered through a 0.02μm tangential flow filtration membrane module at 3℃. The retentate is collected, and the permeate is discarded. Astragalus exosomes of 20-200nm are enriched.

[0108] S5. Ultrafiltration Concentration: Using a 300kDa PES ultrafiltration membrane, exosomes are retained while small molecule impurities are allowed to pass through. The material is concentrated to 1 / 20 of its original volume at 5°C.

[0109] S6. Washing and Filtration: Maintaining stable TMP and CFV parameters from step S5, add sterile PBS buffer (pH 7.4) in three portions, totaling 8 times the volume of the concentrate. After each addition, circulate and filter for 12 minutes, then continue concentrating. Wash and filter until the conductivity of the permeate is consistent with that of PBS (≤20 μS / cm). The final concentrate volume is controlled at 8 mL to obtain pure Astragalus exosomes.

[0110] S7. Purification and Sterilization: The tangential flow filtration concentrate is aseptically filtered sequentially through 0.45μm and 0.22μm PES filter membranes, the filtrate is collected and dispensed into sterile centrifuge tubes.

[0111] S8. Freeze-drying preparation and preservation: Slowly mix the sterile solution with an equal volume of 2 times the concentration of a freeze-drying protectant solution. The protectant used is 5% (w / v) trehalose. Equilibrate at 5°C for 20 minutes. Aliquot the mixed sample solution into freeze-drying vials and rapidly freeze in a -80°C ultra-low temperature freezer or the cold trap of a freeze dryer. Freeze-drying parameters: condenser temperature: ≤-50°C, vacuum degree: 30Pa (0.3mbar), duration: approximately 34 hours, yielding 8.6g of Astragalus exosome powder. When storing the freeze-dried product, it should be filled with high-purity nitrogen and then sealed with a stopper to isolate it from oxygen and moisture. Storage conditions: The freeze-dried powder should be stored in a light-proof, dry environment at ≤-20°C.

[0112] Example 3

[0113] A method for preparing Astragalus exosomes includes the following steps:

[0114] S1. Raw material pretreatment: Select 500g of Astragalus root free from mold and pests, rinse with purified water and air dry. The moisture content of the raw material is ≤12%. After crushing, pass through a 40-mesh sieve, add 5L of physiological saline with pH 7.2, and soak at 5℃ for 12h. Stir for 12min every 6h during the soaking period to obtain Astragalus suspension.

[0115] S2. Cell-wall breaking extraction: The Astragalus suspension was placed in a high-pressure homogenizer and homogenized three times at 100 MPa, with the temperature maintained below 10℃ to prevent degradation of bioactivity. After homogenization, it was allowed to stand at 3℃ for 2 hours to obtain the homogenized suspension.

[0116] S3. Preliminary Purification: The homogenized suspension was centrifuged at 500×g for 15 min at 3℃, and the precipitate (tissue residue) was discarded. The supernatant was then centrifuged at 10000×g for 45 min at 5℃, and the precipitate (organelles) was discarded. After each centrifugation step, the supernatant was carefully transferred to avoid disturbing the precipitate. The final supernatant was filtered through a 0.45μm filter membrane to remove macromolecular impurities, yielding the filtrate; the filtrate was then filtered through a 0.22μm filter membrane.

[0117] S4. Tangential flow filtration concentration: The filtered clear liquid is passed into a tangential flow filtration system equipped with a 0.3μm polyethersulfone (PES) hollow fiber membrane. The material is continuously filtered at 5℃, with the transmembrane pressure (TMP) controlled at 0.05MPa and the tangential flow rate (CFV) at 1.2m / s until the volume of the concentrated liquid is about 50mL. The permeate (containing exosomes and small molecules) is collected, and the retentate (subcellular debris and large protein aggregates) is discarded. The permeate is then continuously filtered through a 0.02μm tangential flow filtration membrane module at 5℃. The retentate is collected, and the permeate is discarded. Astragalus exosomes of 20-300nm are enriched.

[0118] S5. Ultrafiltration Concentration: Using a 200kDa PES ultrafiltration membrane, exosomes are retained while small molecule impurities are allowed to pass through. The material is concentrated to 1 / 15 of its original volume at 3°C.

[0119] S6. Washing and Filtration: Maintaining stable TMP and CFV parameters from step S5, add sterile PBS buffer (pH 7.2) in four portions, five times the volume of the concentrate. After each addition, circulate and filter for 15 minutes, then continue concentrating. Wash and filter until the conductivity of the permeate is consistent with that of PBS (≤20 μS / cm). The final concentrate volume is controlled at 8 mL to obtain a pure concentrate of Astragalus exosomes.

[0120] S7. Purification and sterilization: The concentrated extract of Astragalus exosomes was aseptically filtered through 0.45μm and 0.22μm PES membranes in sequence. The filtrate was collected and dispensed into sterile centrifuge tubes to obtain sterile solution.

[0121] S8. Freeze-drying preparation and preservation: Slowly mix the sterile solution with an equal volume of 2 times the concentration of a freeze-drying protectant solution. The protectant used is 4% (w / v) trehalose. Equilibrate at 3°C ​​for 40 minutes. Aliquot the mixed sample solution into freeze-drying vials and rapidly freeze in a -80°C ultra-low temperature freezer or the cold trap of a freeze dryer. Freeze-drying parameters: condenser temperature: ≤ -50°C, vacuum degree: 50 Pa (0.5 mbar), duration: approximately 30 hours, yielding 8.6 g of Astragalus exosome powder.

[0122] When storing freeze-dried products, high-purity nitrogen should be introduced and the product sealed with a stopper to isolate it from oxygen and moisture. Storage conditions: Freeze-dried powder should be stored in a light-proof, dry environment at ≤ -20℃.

[0123] Comparative Example 1

[0124] A method for preparing Astragalus exosomes, compared with Example 1, except that step S3 is removed, and the rest is the same as Example 1.

[0125] Includes the following steps:

[0126] S1-S2 are the same as in Example 1;

[0127] S3. Tangential Flow Filtration Concentration: The homogenized suspension is passed into a tangential flow filtration system equipped with a 0.15μm polyethersulfone (PES) hollow fiber membrane. The material is continuously filtered at 4℃, with the transmembrane pressure (TMP) controlled at 0.08MPa and the tangential flow rate (CFV) at 1.0m / s until the volume of the concentrated liquid is about 50mL. The permeate (containing exosomes and small molecules) is collected, and the retentate (subcellular debris and large protein aggregates) is discarded. The permeate is then continuously filtered and concentrated through a 0.02μm tangential flow filtration membrane module at 4℃. The retentate is collected, and the permeate is discarded. Astragalus exosomes of 20-150nm are enriched.

[0128] The subsequent steps are the same as steps S5-S8 in Example 1.

[0129] Comparative Example 2

[0130] A method for preparing Astragalus exosomes, which is the same as in Example 1 except that step S4 is removed.

[0131] Steps S1-S3 are the same as in Example 1;

[0132] S4: The filtered clear liquid is filtered through a 100kDa PES ultrafiltration membrane to retain exosomes while allowing small molecule impurities to pass through. The material is concentrated to 1 / 10 of its original volume at 4°C.

[0133] The subsequent steps are the same as steps S6-S8 in Example 1.

[0134] Comparative Example 3

[0135] A method for preparing Astragalus exosomes, compared with Example 1, step S5 is removed, and the rest is the same as Example 1.

[0136] Steps S1-S4 are the same as in Example 1;

[0137] S5. Washing and Filtration: Maintaining stable TMP and CFV parameters from step S4, add sterile PBS buffer (pH 7.2) in four portions, with a total volume six times the volume of the permeate from step S4. After each addition, circulate and filter for 15 minutes, then continue concentrating. Wash and filter until the conductivity of the permeate is consistent with that of PBS (≤20 μS / cm). The final volume of the concentrated solution is controlled at 8 mL to obtain a pure concentrated solution of Astragalus exosomes.

[0138] The subsequent steps are the same as steps S7-S8 in Example 1.

[0139] Comparative Example 4

[0140] A method for preparing Astragalus exosomes, compared with Example 1, step S6 is removed, and the rest is the same as Example 1.

[0141] Steps S1-S5 are the same as in Example 1;

[0142] S6. Purification and sterilization: The concentrate obtained in step S5 is aseptically filtered through 0.45μm and 0.22μm PES filter membranes in sequence. The filtrate is collected and dispensed into sterile centrifuge tubes to obtain sterile solution.

[0143] S7. Freeze-drying preparation and preservation: Slowly mix the sterile solution with an equal volume of 2 times the concentration of a freeze-drying protectant solution. The protectant used is 4% (w / v) trehalose. Equilibrate at 4°C for 30 minutes. Aliquot the mixed sample solution into freeze-drying vials and rapidly freeze them in a -80°C ultra-low temperature freezer or the cold trap of a freeze dryer. Freeze-drying parameters: condenser temperature: ≤ -50°C, vacuum degree: 50 Pa (0.5 mbar), duration: approximately 30 hours, yielding 8.6 g of Astragalus exosome dry powder.

[0144] Comparative Example 5

[0145] Astragalus exosomes were prepared according to the preparation method described in the example section

[0043] of Chinese patent application CN11933180 A.

[0146] Test Example 1

[0147] Quality testing of Astragalus exosomes:

[0148] (1) Appearance and properties:

[0149] The Astragalus exosome lyophilized powders prepared in Examples 1-3 and Comparative Examples 1-5 were pale yellow, loose powders without lumps; when reconstituted with physiological saline to 1 mg / mL, the solution was clear and transparent, with no visible impurities. Electron micrographs of Examples 1 and 2 are shown below. Figures 1-2 As shown.

[0150] (2) Particle size distribution: The particle size of Examples 1-3 and Comparative Examples 1-5 was detected by NTA, and the results are shown in Table 1 below:

[0151] Table 1

[0152]

[0153] Test Example 2

[0154] Validation of a zebrafish lung cancer (A549 cell transplantation) model:

[0155] 2.1 Animal husbandry and handling:

[0156] Adult zebrafish were housed in a recirculating aquaculture system at a maintained temperature of 28°C, with a photocycle of 14 hours of light / 10 hours of darkness, and were fed three times daily. To obtain embryos, sexually mature zebrafish were paired at a 1:1 female-to-male ratio in the evening and separated by a baffle. The baffle was removed within one hour of the start of the next photocycle to allow for natural spawning. The collected embryos were placed in a 1×E3 solution (10cm diameter petri dish) containing 0.3 ppm methylene blue and cultured in a 28.5°C artificial climate chamber (14h / 10h, light / dark) until the experimental treatment stage (2 days post-fertilization, i.e., 2 days post-fertilization).

[0157] 2.2 Instruments, Consumables and Reagents

[0158] Analytical balance (ME204, METTLERTOLEDO, Switzerland);

[0159] Motorized fluorescence zoom microscope (AxioZoom.V16, ZEISS, Germany);

[0160] Stereo microscope (Stemi 508, ZEISS, Germany);

[0161] Orange-red fluorescent probe for cell membrane (CM-Dil, Lot: KS378662, Shanghai Yuanye Biotechnology Co., Ltd., China);

[0162] Methylcellulose (Lot: E2218153, Aladdin, China);

[0163] RNA extraction kit (Lot:7E2140C5, Novizan, China); reverse transcription kit (Lot:7E0650B4, Novizan, China); qPCR mix (Lot:7E0790D4, Novizan, China);

[0164] Dimethyl sulfoxide (DMSO, Lot: BCCD8942, Sigma, Switzerland).

[0165] 2.3 Test Methods

[0166] Two-day-old (2 dpf) zebrafish were randomly selected for the experiment in 6-well plates (3 mL / well), with 30 fish per well. The plates included a model control group, a methotrexate group, Examples 1-3, and Comparative Examples 1-5. Each well was injected with 200 A549 tumor cells to establish a lung cancer model. After 24 h of incubation, the methotrexate group was treated with 200 μM methotrexate solution, while the astragalus exosome groups were treated with different concentrations of astragalus exosome solution. After 48 h of treatment, images were captured under a fluorescence microscope, and the antitumor (lung cancer) efficacy of astragalus exosomes was evaluated by statistically analyzing the fluorescence intensity of zebrafish lung cancer cells.

[0167] .

[0168] Statistical analysis was performed using GraphPad Prism 8.0 software. Quantitative data are expressed as mean ± standard error (mean ± SEM). Independent samples t-tests were used to compare two groups. A p-value < 0.05 was considered statistically significant. The experimental results are shown in Table 2.

[0169] Table 2

[0170]

[0171] Note: Compared with the model control group, This represents p < 0.001; compared with Example 1 group, # This means p < 0.05. ## This means p < 0.01.

[0172] As shown in Table 2, the Astragalus exosomes prepared in this invention have significant anti-tumor (lung cancer) effects.

[0173] Test Example 3

[0174] Validation of the zebrafish breast cancer (MCF-7 cell transplantation) model:

[0175] 3.1 Animal husbandry and handling:

[0176] Adult zebrafish were housed in a recirculating aquaculture system at a maintained temperature of 28°C, with a photocycle of 14 hours of light / 10 hours of darkness, and were fed three times daily. To obtain embryos, sexually mature zebrafish were paired at a 1:1 female-to-male ratio in the evening and separated by a baffle. The baffle was removed within one hour of the start of the next photocycle to allow for natural spawning. The collected embryos were placed in a 1×E3 solution (10cm diameter petri dish) containing 0.3 ppm methylene blue and cultured in a 28.5°C artificial climate chamber (14h / 10h, light / dark) until the experimental treatment stage (2 days post-fertilization, i.e., 2 days post-fertilization).

[0177] 3.2 Instruments, Consumables and Reagents

[0178] Analytical balance (ME204, METTLERTOLEDO, Switzerland);

[0179] Motorized fluorescence zoom microscope (AxioZoom.V16, ZEISS, Germany);

[0180] Stereo microscope (Stemi 508, ZEISS, Germany);

[0181] Orange-red fluorescent probe for cell membrane (CM-Dil, Lot: KS378662, Shanghai Yuanye Biotechnology Co., Ltd., China);

[0182] Methylcellulose (Lot: E2218153, Aladdin, China);

[0183] RNA extraction kit (Lot:7E2140C5, Novizan, China); reverse transcription kit (Lot:7E0650B4, Novizan, China); qPCR mix (Lot:7E0790D4, Novizan, China);

[0184] Dimethyl sulfoxide (DMSO, Lot: BCCD8942, Sigma, Switzerland).

[0185] 3.3 Test Methods

[0186] Two days post-fertilization (2 dpf) zebrafish were randomly selected and subjected to experiments in 6-well plates (3 mL / well), with 30 fish per well. A model control group, a tamoxifen group, Examples 1-3, and Comparative Examples 1-5 were all established by injecting 200 MCF-7 tumor cells. After 24 h of incubation, the tamoxifen group was treated with 2 μM tamoxifen solution, and the Astragalus exosome groups were treated with different concentrations of Astragalus exosome solution. After 48 h of treatment, images were captured under a fluorescence microscope, and the antitumor (breast cancer) efficacy of Astragalus exosomes was evaluated by statistically analyzing the fluorescence intensity of zebrafish breast cancer cells.

[0187] .

[0188] Statistical analysis was performed using GraphPad Prism 8.0 software. Quantitative data are expressed as mean ± standard error (mean ± SEM). Independent samples t-tests were used to compare two groups. p < 0.05 was considered statistically significant. The experimental results are shown in Table 3.

[0189] Table 3

[0190]

[0191] Note: Compared with the model control group, This means p < 0.05. This represents p < 0.01; compared with Example 1 group, # This means p < 0.05. ## This means p < 0.01.

[0192] As shown in Table 3, the Astragalus exosomes prepared in this invention have significant anti-tumor (breast cancer) effects.

[0193] Test Example 4

[0194] Further experiments were conducted using the Astragalus exosomes prepared in Example 1, which showed the best results:

[0195] 4.1 Animal husbandry and handling:

[0196] Adult zebrafish were housed in a recirculating aquaculture system at a maintained temperature of 28°C, with a photocycle of 14 hours of light / 10 hours of darkness, and were fed three times daily. To obtain embryos, sexually mature zebrafish were paired at a 1:1 female-to-male ratio in the evening and separated by a baffle. The baffle was removed within one hour of the start of the next photocycle to allow for natural spawning. The collected embryos were placed in a 1×E3 solution (10cm diameter petri dish) containing 0.3 ppm methylene blue and cultured in a 28.5°C artificial climate chamber (14h / 10h, light / dark) until the experimental treatment stage (2 days post-fertilization, i.e., 2 days post-fertilization).

[0197] 4.2 Instruments, Consumables and Reagents

[0198] Analytical balance (ME204, METTLERTOLEDO, Switzerland);

[0199] Motorized fluorescence zoom microscope (AxioZoom.V16, ZEISS, Germany);

[0200] Stereo microscope (Stemi 508, ZEISS, Germany);

[0201] Orange-red fluorescent probe for cell membrane (CM-Dil, Lot: KS378662, Shanghai Yuanye Biotechnology Co., Ltd., China);

[0202] Methylcellulose (Lot: E2218153, Aladdin, China);

[0203] RNA extraction kit (Lot:7E2140C5, Novizan, China); reverse transcription kit (Lot:7E0650B4, Novizan, China); qPCR mix (Lot:7E0790D4, Novizan, China);

[0204] Dimethyl sulfoxide (DMSO, Lot: BCCD8942, Sigma, Switzerland).

[0205] 4.3 Experimental groups: normal control group, model control group (200 tumor cells), and Example 1 group (hereinafter referred to as Astragalus exosome group, with 5 concentration gradients: 2000 μg / mL, 1000 μg / mL, 500 μg / mL, 250 μg / mL, 125 μg / mL), for a total of 7 groups.

[0206] 4.4 Effects on normal zebrafish

[0207] Treatment method: Zebrafish 3 days post-fertilization (3dpf) were randomly selected and subjected to the experiment in 6-well plates (3 mL / well), with 30 fish per well. Different concentrations of Astragalus exosome solution were added to the normal control group and the Astragalus exosome group, respectively. After treatment for 48 hours, images were taken under a microscope to observe the zebrafish phenotype. The results are shown in Table 4 below:

[0208] Table 4

[0209]

[0210] The results showed that 1000 μg / mL Astragalus exosomes had no effect on normal zebrafish after 48 hours of exposure. See details. Figure 3 and Figure 4 .

[0211] 4.5 Antitumor Trial

[0212] The antitumor effects of the three concentrations (1000 μg / mL, 500 μg / mL, and 250 μg / mL) in Example 1 were verified according to the methods described in Examples 3 and 4. The results are shown in Tables 5 (lung cancer) and 6 (breast cancer).

[0213] Table 5

[0214]

[0215] Note: Compared with the model control group, This means p < 0.001.

[0216] Table 6

[0217]

[0218] Note: Compared with the model control group, This means p < 0.001.

[0219] Figure 5 Typical fluorescence intensity diagram of zebrafish lung cancer cells after treatment with Astragalus membranaceus exosomes. Figure 6 Corresponding to the fluorescence intensity (pixels) of zebrafish lung cancer after treatment with Astragalus exosomes, as shown in Tables 5-6 and Figures 5-6 It can be seen that the Astragalus exosomes prepared in Example 1 have significant anti-tumor effects (lung cancer and breast cancer) at three concentrations of 1000 μg / mL, 500 μg / mL and 250 μg / mL.

[0220] 4.6 Anti-tumor effect (relative gene expression level)

[0221] Zebrafish 2 days post-fertilization (2 dpf) were randomly selected and subjected to experiments in 6-well plates (3 mL / well), with 30 fish per well. Both the model control group and the Astragalus exosome group were injected with 200 A549 tumor cells to establish a lung cancer model. After 24 h of incubation, different concentrations of Astragalus exosome solution were added to the Astragalus exosome group. After 48 h of treatment, total RNA was extracted from the zebrafish and reverse transcribed into cDNA. The expression levels of the internal reference gene β-actin and the gene il6 were detected using qPCR to evaluate the antitumor effect of Astragalus exosomes. Data analysis was performed using GraphPad Prism 8.0 statistical software.

[0222] .

[0223] Quantitative data are expressed as mean ± standard error (mean ± SEM). Independent samples t-tests were used to compare the two groups. p < 0.05 was considered statistically significant. Gene primer sequences are shown in Table 7, and experimental results are shown in Table 8.

[0224] Table 7

[0225]

[0226] Table 8

[0227]

[0228] Compared with the model control group, This represents p < 0.05. This means p < 0.001.

[0229] As shown in Table 8, the results of the experiment indicate that the relative expression levels of gene il6 were reduced by 23.73%, 43.25%, and 50.66% at concentrations of 250 μg / mL, 500 μg / mL, and 1000 μg / mL, respectively, revealing that astragalus exosomes have anti-tumor effects.

[0230] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing Astragalus exosomes, characterized in that, Includes the following steps: S1： Pretreatment: Astragalus root was soaked in physiological saline to obtain Astragalus suspension; S2: Cell wall disruption extraction: Astragalus suspension is homogenized 1-3 times under 80-100MPa pressure to obtain homogenized suspension; S3: Preliminary purification: Centrifuge the homogenized suspension at a relative centrifugal force of 500-10000×g, a temperature of 3-5℃, a time of 15-45min, and repeat 1-3 times; obtain supernatant 1, which is then filtered through a filter membrane with a pore size of 0.22-0.45μm; the filtration is repeated 1-3 times. The filtration process includes the following steps: supernatant 1 is passed through a 0.45μm filter membrane to remove macromolecular impurities, resulting in filtrate; The filtrate was then passed through a 0.22 μm filter membrane to obtain supernatant 2; S4: Tangential flow filtration concentration: S4-1: The supernatant 2 is continuously filtered and concentrated at 3-5℃ through a 0.15-0.3μm tangential flow filtration membrane module. The permeate is collected and the retained material is discarded. S4-2: The permeate is then continuously filtered and concentrated at 3-5℃ through a 0.015-0.025μm tangential flow filtration membrane module. The retentate is collected and the permeate is discarded. S5: Ultrafiltration Concentration: The retentate is filtered through a 100-300kDa ultrafiltration membrane to retain exosomes and then concentrated to obtain a concentrated solution; S6: Washing and filtration: Use PBS buffer as the washing solution, wash and filtration the concentrated solution 2-5 times, concentrate, and obtain the pure concentrated solution of Astragalus exosomes; S7: Purification and sterilization: Aseptic filtration of the pure extract of Astragalus exosomes yields the product.

2. The preparation method according to claim 1, characterized in that, In step S2, the homogenization pressure is 80-100 MPa, the number of times is 1-3, and the temperature is <10℃.

3. The preparation method according to claim 1, characterized in that, In steps S4-1 and S4-2, the transmembrane pressure of the continuous filtration is 0.05-0.2 MPa, and the tangential flow velocity is 1.0-1.5 m / s.

4. The preparation method according to claim 1, characterized in that, In step S5, the volume is concentrated to 1 / 10-1 / 20 of the original volume.

5. The preparation method according to claim 1, characterized in that, In step S6, the washing and filtration includes the following steps: maintaining the transmembrane pressure at 0.1-0.15 MPa and the tangential flow rate at 1.2-1.8 m / s, adding sterile PBS buffer to the concentrate, the volume of sterile PBS buffer being 5-8 times that of the concentrate, adding it in 3-4 portions, and circulating and filtering for 10-15 min after each addition.

6. The preparation method according to claim 1, characterized in that, In step S7, the aseptic filtration includes the following steps: the concentrated extract of Astragalus exosomes is passed sequentially through filter membranes of 0.4-0.5μm and 0.2-0.3μm, and the filtrate is collected to obtain Astragalus exosomes.

7. Astragalus exosomes prepared by the preparation method according to any one of claims 1-6.

8. The use of the Astragalus exosomes according to claim 7 in the preparation of drugs for treating lung cancer.

9. The use of the Astragalus exosomes according to claim 7 in the preparation of an anti-breast cancer drug.

Images