Preparation method of plant exosome compound based on ultrapure technology

Abstract

The invention belongs to the technical field of biotechnology and cell preparation, and particularly relates to a preparation method of a plant exosome compound based on an ultra-pure technology, which comprises the following steps: S1, treating plant tissues by adopting a low-temperature enzyme-assisted wall breaking technology to prepare plant tissue homogenate; s2, carrying out primary clarification on the homogenate obtained in the step S1, and removing large-particle impurities; s3, carrying out microfiltration pretreatment on the clarified liquid obtained in S2 through a polyether sulfone membrane; s4, carrying out tangential flow ultrafiltration on the filtrate obtained in S3 to remove macromolecular impurities; s5, performing tangential flow nanofiltration on the filtrate obtained in the step S4 to enrich exosomes; s6, performing iodixanol and sucrose mixed density gradient separation on the enrichment liquid obtained in S5, and collecting exosomes in corresponding density intervals; s7, the exosome obtained in the step S6 is subjected to buffer solution replacement, and the density gradient agent is removed; and S8, adding a low-temperature freeze-drying protective agent into the plant exosome obtained in the S7 to prepare the plant exosome compound.

Description

Technical Field

[0001] The invention belongs to the fields of biotechnology and cell preparation technology, specifically relating to a method for preparing plant exosome complexes based on ultrapure technology, and particularly to a method for preparing plant exosome complexes by combining tangential flow filtration technology with density gradient separation technology and adding a specific low-temperature freeze-drying protectant. Background Technology

[0002] Recent studies have discovered that most organisms in nature (including animals, plants, and certain bacteria) can secrete nanoscale vesicle-like structures encased in a phospholipid bilayer, known as exosomes. Plant exosomes, as a novel bioactive ingredient, show great promise for applications in beauty, skincare, functional foods, and drug delivery systems.

[0003] Currently, the isolation and extraction technology of plant exosomes is mainly based on ultracentrifugation. For example, Chinese patent CN114540268 B discloses "a method for preparing plant exosome complexes based on ultrapure solid membrane technology". This method uses a combination of ultracentrifugation and multiple filtration to obtain plant exosomes, and adds solid membrane components (mannitol, trehalose, and maltodextrin) to prepare plant exosome complexes. However, this technology has certain limitations: First, the high shear force generated during ultracentrifugation can cause some damage to the exosome structure, affecting the integrity and activity of the exosomes; second, the process of removing impurities and cell debris by multiple differential centrifugations is not only time-consuming, but also further increases the damage to the exosomes; third, the purity of the obtained plant exosomes is relatively low, and the particle size is not uniform; finally, to obtain plant exosomes with higher purity, sucrose density gradient centrifugation is usually required for purification, which is complex, time-consuming, and results in significant exosome loss.

[0004] Therefore, there is an urgent need to develop a method for preparing plant exosome complexes that can simultaneously address the issues of exosome integrity, purity, uniformity, and stability. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing plant exosome complexes based on ultrapure technology. This method combines tangential flow filtration technology with iodixanol / sucrose density gradient separation technology and adds a specific low-temperature freeze-drying protectant to solve the technical problems of low purity, uneven particle size, and unstable activity of plant exosomes in the prior art.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method for preparing plant exosome complexes based on ultrapure technology, the method comprising the following steps:

[0008] S1: Plant tissue homogenate was prepared by processing plant tissue using low-temperature enzyme-assisted cell disruption technology.

[0009] S2: Perform primary clarification on the homogenate obtained in S1 to remove large particulate impurities;

[0010] S3: The clarified liquid obtained in S2 is pretreated by microfiltration through a polyethersulfone membrane;

[0011] S4: The filtrate obtained in S3 is subjected to tangential flow ultrafiltration to remove macromolecular impurities;

[0012] S5: The filtrate obtained in S4 is enriched with exosomes by tangential flow nanofiltration;

[0013] S6: The enriched solution obtained in S5 was subjected to density gradient separation by mixing iodixanol and sucrose, and exosomes in the corresponding density range were collected.

[0014] S7: Replace the exosomes obtained in S6 with buffer solution to remove the density gradient agent;

[0015] S8: Add a low-temperature freeze-drying protectant to the plant exosomes obtained in S7 to prepare a plant exosome complex.

[0016] Preferably, the low-temperature enzyme-assisted cell disruption technology in S1 includes:

[0017] Fresh plant tissue was washed, drained, and chopped. It was then mixed with extraction buffer at a ratio of 1:2 (plant weight to extraction buffer). The extraction buffer consisted of 20 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EDTA, and 0.1-0.25% cellulase, 0.05-0.15% pectinase, and 0.025-0.075% hemicellulase. The mixture was stirred at 60-120 rpm for 8-12 hours at 4°C. After stirring, the mixture was homogenized for 3-5 minutes to obtain a plant tissue homogenate.

[0018] Preferably, the primary clarification method in S2 is as follows: the plant tissue homogenate obtained in S1 is centrifuged at 3,000-5,000g for 30 minutes at 4°C; the supernatant is collected to obtain a preliminarily clarified plant homogenate.

[0019] Preferably, the microfiltration pretreatment method in S3 is as follows: the clarified liquid obtained in S2 is microfiltered through a 5μm polyethersulfone membrane, the operating temperature is controlled at 4-8°C, and the membrane flux is controlled at 30-50 L / m² / h.

[0020] Preferably, the tangential flow ultrafiltration method in S4 is as follows: the filtrate obtained in S3 is ultrafiltered through a polyethersulfone hollow fiber tangential flow filtration system with a molecular weight cutoff of 500 kDa; the ultrafiltration operation temperature is controlled at 4-8°C, the transmembrane pressure difference is controlled at 0.1-0.2 bar, and the tangential flow rate is controlled at 100-150 cm / s; constant volume ultrafiltration is performed to concentrate the volume to 1 / 5-1 / 10 of the original volume; a two-step dialysis elution method is used, with 5 times the volume of 20 mM HEPES pH 7.4 and 150 mM NaCl buffer for dialysis elution; the filtrate is collected to obtain a solution rich in exosomes.

[0021] Preferably, the tangential flow nanofiltration method in S5 is as follows: the filtrate obtained in S4 is subjected to nanofiltration through a polyethersulfone hollow fiber tangential flow filtration system with a molecular weight cutoff of 100 kDa; the nanofiltration operation temperature is controlled at 4-8°C, the transmembrane pressure difference is controlled at 0.05-0.15 bar, and the tangential flow rate is controlled at 80-120 cm / s; the volume is concentrated to 1 / 10-1 / 20 of the original volume; the filtrate is collected to obtain a concentrated exosome solution.

[0022] Preferably, the density gradient separation method in S6 is as follows: a mixed density gradient solution of iodixanol and sucrose is prepared and divided into 5 layers: the first layer is 5% iodixanol and 5% sucrose, the second layer is 10% iodixanol and 10% sucrose, the third layer is 20% iodixanol and 15% sucrose, the fourth layer is 30% iodixanol and 20% sucrose, and the fifth layer is 40% iodixanol and 25% sucrose; each layer of density gradient solution is added sequentially from bottom to top in an ultracentrifuge tube, 2 mL for each layer; finally, the concentrated exosome solution obtained in S5 is added to the top layer of the density gradient; the mixture is centrifuged at 100,000 g for 16 hours at 4°C; and the exosome layer with a density of approximately 1.15-1.19 g / mL is collected.

[0023] Preferably, the buffer replacement method in S7 is as follows: the exosome layer obtained in S6 is replaced with buffer through an ultrafiltration tube with a molecular weight cutoff of 10 kDa; 10 volumes of 20 mM HEPES pH 7.4, 150 mM NaCl buffer are added, and the mixture is centrifuged at 3,000 g for 30 minutes at 4°C; the filtrate is discarded, and the above steps are repeated three times to completely remove iodixanol and sucrose; the retentate is collected to obtain purified plant exosomes.

[0024] Preferably, the low-temperature freeze-drying protectant in S8 is added in the following mass fraction ratios: 1.5-2.8% sodium carboxymethyl cellulose, 2.2-3.5% sodium alginate, 3.5-4.5% polyvinylpyrrolidone K30, and 0.5-1.5% hydroxypropyl-β-cyclodextrin.

[0025] Preferably, the method for preparing plant exosome complex dry powder in step S8 is as follows: the plant exosome complex with added low-temperature freeze-drying protectant is dispensed into freeze-drying bottles; pre-frozen at -80°C for 2 hours; freeze-dried at -55°C for 48 hours to obtain plant exosome complex dry powder. Detailed Implementation

[0026] The present invention will be further described in detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0027] Unless otherwise specified, all raw materials and equipment used in this invention can be obtained through conventional commercial channels. The enzyme preparations (cellulase, pectinase, and hemicellulase) used in this invention were purchased from Sigma-Aldrich; iodixanol was purchased from GE Healthcare's Optiprep™ product; the polyethersulfone (PES) membrane and tangential flow filtration system were purchased from Millipore; sodium carboxymethyl cellulose (model: C5678), sodium alginate (model: A2158), polyvinylpyrrolidone K30 (model: PVP40), and hydroxypropyl-β-cyclodextrin (model: H107) were all purchased from Sigma-Aldrich; other reagents were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd.

[0028] Example 1: Preparation of Citrus Exosome Complex

[0029] This embodiment provides a method for preparing a citrus exosome complex based on ultrapure technology, specifically including the following steps:

[0030] (1) Low-temperature enzyme-assisted cell wall disruption: Take 500 g of fresh citrus peel, wash and drain it, and cut it into small pieces of about 0.5 cm × 0.5 cm. Add extraction buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.2% cellulase, 0.1% pectinase and 0.05% hemicellulase) at a ratio of 1:2, place it in a 4°C environment, and stir at 80 rpm for 10 hours. After stirring, use a tissue homogenizer (IKA T25 basic type) to homogenize at 8,000 rpm for 4 minutes to obtain citrus tissue homogenate.

[0031] (2) Primary clarification: The obtained citrus tissue homogenate was transferred to a centrifuge tube and centrifuged at 4°C for 30 minutes at a centrifugal force of 4,000 g (using a Beckman Coulter Allegra X-15R centrifuge). The supernatant was carefully collected to obtain a preliminary clarified citrus homogenate of about 1,500 mL.

[0032] (3) Microfiltration pretreatment: The clarified citrus homogenate was microfiltered through a 5 μm polyethersulfone membrane at an operating temperature of 6°C and a membrane flux of 40 L / m² / h. Approximately 1,450 mL of filtrate was collected for later use.

[0033] (4) Tangential flow ultrafiltration: The microfiltration filtrate was ultrafiltered through a polyethersulfone hollow fiber tangential flow filtration system with a molecular weight cutoff of 500 kDa (using a Millipore Pellicon XL device). The ultrafiltration operation temperature was 6°C, the transmembrane pressure difference was 0.15 bar, and the tangential flow rate was 120 cm / s. Constant volume ultrafiltration was performed to concentrate the solution to 1 / 8 of its original volume (approximately 180 mL). Subsequently, the solution was eluted by dialyzing with 5 volumes of 20 mM HEPES pH 7.4, 150 mM NaCl buffer (approximately 900 mL) to remove small molecule impurities. Approximately 175 mL of the filtrate was collected to obtain a solution rich in exosomes.

[0034] (5) Tangential flow nanofiltration: The ultrafiltration filtrate was subjected to nanofiltration through a polyethersulfone hollow fiber tangential flow filtration system with a molecular weight cutoff of 100 kDa. The nanofiltration operating temperature was 6°C, the transmembrane pressure difference was 0.1 bar, and the tangential flow rate was 100 cm / s. The solution was concentrated to 1 / 15 of its original volume (approximately 12 mL). The retentate was collected to obtain a concentrated citrus exosome solution.

[0035] (6) Density gradient separation: Prepare a mixed density gradient solution of iodixanol and sucrose, dividing it into 5 layers: Layer 1: 5% iodixanol and 5% sucrose; Layer 2: 10% iodixanol and 10% sucrose; Layer 3: 20% iodixanol and 15% sucrose; Layer 4: 30% iodixanol and 20% sucrose; Layer 5: 40% iodixanol and 25% sucrose. Carefully add each layer of density gradient solution sequentially from bottom to top into an ultracentrifuge tube, 2 mL for each layer. Finally, carefully add the concentrated citrus exosome solution (approximately 2 mL) to the top layer of the density gradient. Centrifuge at 100,000 g for 16 hours at 4°C (using a Beckman Coulter Optima XE-90 ultracentrifuge). After centrifugation, each interface was carefully separated from top to bottom, and about 1.5 mL of the exosome layer with a density of about 1.17 g / mL (located between the 20% iodixanol + 15% sucrose layer and the 30% iodixanol + 20% sucrose layer) was collected.

[0036] (7) Buffer replacement: Transfer the collected citrus exosome layer to an ultrafiltration tube (Millipore Amicon Ultra-15) with a molecular weight cutoff of 10 kDa, and add 10 volumes of 20 mM HEPES pH 7.4, 150 mM NaCl buffer (approximately 15 mL). Centrifuge at 3,000 g for 30 minutes at 4°C. Discard the filtrate and repeat the above steps three times to completely remove iodixanol and sucrose. Collect approximately 1.2 mL of the retentate to obtain purified citrus exosomes.

[0037] (8) Adding cryoprotectants: The concentration of the obtained citrus exosomes was determined to be 2.5 mg / mL (using the BCA protein quantification kit). Cryoprotectants were added according to the following mass fraction ratios: 2% sodium carboxymethyl cellulose, 3% sodium alginate, 4% polyvinylpyrrolidone K30, and 1% hydroxypropyl-β-cyclodextrin. Specifically, 24 mg of sodium carboxymethyl cellulose, 36 mg of sodium alginate, 48 mg of polyvinylpyrrolidone K30, and 12 mg of hydroxypropyl-β-cyclodextrin were added to 1.2 mL of the citrus exosome solution. The mixture was gently stirred until the protectants were completely dissolved, yielding a liquid formulation of the citrus exosome complex.

[0038] (9) Preparation of dry powder: The liquid formulation was dispensed into pre-cooled lyophilization bottles, approximately 0.5 mL per bottle. The bottles were pre-frozen in an ultra-low temperature freezer at -80°C for 2 hours. Subsequently, the lyophilization bottles were transferred to a lyophilizer (Christ Alpha 2-4 LSC lyophilizer) and lyophilized at -55°C for 48 hours to obtain the citrus exosome complex dry powder.

[0039] Example 2: Preparation of Green Tea Exosome Complex

[0040] This embodiment provides a method for preparing a green tea exosome complex based on ultrapure technology, specifically including the following steps:

[0041] (1) Low-temperature enzyme-assisted cell wall disruption: Take 300 g of fresh green tea leaves, wash and drain them, then chop them. Add extraction buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.15% cellulase, 0.08% pectinase and 0.04% hemicellulase) at a ratio of 1:2, place in a 4°C environment, and stir at 70 rpm for 12 hours. After stirring, use a tissue homogenizer to homogenize at 7,000 rpm for 5 minutes to obtain green tea tissue homogenate.

[0042] (2) Primary clarification: The obtained green tea tissue homogenate was transferred to a centrifuge tube and centrifuged at 3,500 g for 30 minutes at 4°C. The supernatant was collected to obtain a preliminarily clarified green tea homogenate, about 900 mL.

[0043] (3) Microfiltration pretreatment: The clarified green tea homogenate was microfiltered through a 5 μm polyethersulfone membrane. The operating temperature was controlled at 5°C and the membrane flux was controlled at 35 L / m² / h. Approximately 860 mL of filtrate was collected for later use.

[0044] (4) Tangential flow ultrafiltration: The microfiltration filtrate was ultrafiltered through a polyethersulfone hollow fiber tangential flow filtration system with a molecular weight cutoff of 500 kDa. The ultrafiltration operating temperature was 5°C, the transmembrane pressure difference was 0.12 bar, and the tangential flow rate was 110 cm / s. Constant volume ultrafiltration was performed to concentrate the solution to 1 / 6 of its original volume (approximately 145 mL). Subsequently, the solution was eluted by dialyzing with 5 volumes of 20 mM HEPES pH 7.4, 150 mM NaCl buffer (approximately 725 mL). Approximately 140 mL of the filtrate was collected to obtain a solution rich in exosomes.

[0045] (5) Tangential flow nanofiltration: The ultrafiltration filtrate was subjected to nanofiltration through a polyethersulfone hollow fiber tangential flow filtration system with a molecular weight cutoff of 100 kDa. The nanofiltration operating temperature was 5°C, the transmembrane pressure difference was 0.08 bar, and the tangential flow rate was 90 cm / s. The solution was concentrated to 1 / 12 of its original volume (approximately 12 mL). The retentate was collected to obtain a concentrated green tea exosome solution.

[0046] (6) Density gradient separation: Iodixanol and sucrose mixed density gradient solutions were prepared using the same method as in Example 1, and density gradient separation was performed. Exosome layer with a density of approximately 1.16 g / mL, approximately 1.3 mL, was collected.

[0047] (7) Buffer replacement: Buffer replacement was performed using the same method as in Example 1 to obtain approximately 1.0 mL of purified green tea exosomes.

[0048] (8) Addition of cryoprotectant: The concentration of green tea exosomes obtained was determined to be 2.2 mg / mL. Cryoprotectant was added according to the following mass fraction ratios: 2.5% sodium carboxymethyl cellulose, 2.5% sodium alginate, 3.5% polyvinylpyrrolidone K30, and 1.5% hydroxypropyl-β-cyclodextrin. Specifically, 25 mg of sodium carboxymethyl cellulose, 25 mg of sodium alginate, 35 mg of polyvinylpyrrolidone K30, and 15 mg of hydroxypropyl-β-cyclodextrin were added to 1.0 mL of green tea exosome solution. The mixture was gently stirred until the protectant was completely dissolved, yielding a liquid formulation of the green tea exosome complex.

[0049] (9) Preparation of dry powder: freeze-drying was carried out in the same way as in Example 1 to obtain green tea exosome complex dry powder.

[0050] Example 3: Preparation of grape exosome complex

[0051] This embodiment provides a method for preparing grape exosome complexes based on ultrapure technology, specifically including the following steps:

[0052] (1) Low-temperature enzyme-assisted cell wall disruption: Take 600 g of fresh grapes, wash and drain them, and remove the seeds. Add extraction buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.25% cellulase, 0.12% pectinase and 0.06% hemicellulase) at a ratio of 1:2, and place in a 4°C environment. Stir at 90 rpm for 9 hours. After stirring, use a tissue homogenizer to homogenize at 9,000 rpm for 3 minutes to obtain grape tissue homogenate.

[0053] (2) Primary clarification: The obtained grape tissue homogenate was transferred to a centrifuge tube and centrifuged at 4°C and 4,500 g for 30 minutes. The supernatant was collected to obtain a preliminarily clarified grape homogenate of about 1,800 mL.

[0054] (3) Microfiltration pretreatment: The clarified grape homogenate was microfiltered through a 5 μm polyethersulfone membrane at an operating temperature of 7°C and a membrane flux of 45 L / m² / h. Approximately 1,720 mL of filtrate was collected for later use.

[0055] (4) Tangential flow ultrafiltration: The microfiltration filtrate was ultrafiltered through a polyethersulfone hollow fiber tangential flow filtration system with a molecular weight cutoff of 500 kDa. The ultrafiltration operation temperature was 7°C, the transmembrane pressure difference was 0.18 bar, and the tangential flow rate was 140 cm / s. Constant volume ultrafiltration was performed to concentrate the solution to 1 / 10 of its original volume (approximately 172 mL). Subsequently, the solution was eluted by dialyzing with 5 volumes of 20 mM HEPES pH 7.4, 150 mM NaCl buffer (approximately 860 mL). Approximately 165 mL of the filtrate was collected to obtain a solution rich in exosomes.

[0056] (5) Tangential flow nanofiltration: The ultrafiltration filtrate was subjected to nanofiltration through a polyethersulfone hollow fiber tangential flow filtration system with a molecular weight cutoff of 100 kDa. The nanofiltration operating temperature was 7°C, the transmembrane pressure difference was 0.12 bar, and the tangential flow rate was 110 cm / s. The solution was concentrated to 1 / 15 of its original volume (approximately 11 mL). The retentate was collected to obtain a concentrated grape exosome solution.

[0057] (6) Density gradient separation: Iodixanol and sucrose mixed density gradient solutions were prepared using the same method as in Example 1, and density gradient separation was performed. Exosome layer with a density of approximately 1.18 g / mL was collected, approximately 1.8 mL.

[0058] (7) Buffer replacement: Buffer replacement was performed using the same method as in Example 1 to obtain approximately 1.5 mL of purified grape exosomes.

[0059] (8) Addition of cryoprotectant: The concentration of grape exosomes obtained was determined to be 3.1 mg / mL. The cryoprotectant was added according to the following mass fraction ratios: 1.5% sodium carboxymethyl cellulose, 3.5% sodium alginate, 4.5% polyvinylpyrrolidone K30, and 0.5% hydroxypropyl-β-cyclodextrin. Specifically, 22.5 mg of sodium carboxymethyl cellulose, 52.5 mg of sodium alginate, 67.5 mg of polyvinylpyrrolidone K30, and 7.5 mg of hydroxypropyl-β-cyclodextrin were added to 1.5 mL of grape exosome solution. The mixture was gently stirred until the protectant was completely dissolved, yielding a liquid formulation of the grape exosome complex.

[0060] (9) Preparation of dry powder: freeze-drying was carried out in the same way as in Example 1 to obtain grape exosome complex dry powder.

[0061] Example 4: Preparation of onion exosome complex

[0062] This embodiment provides a method for preparing an onion exosome complex based on ultrapure technology, specifically including the following steps:

[0063] (1) Low-temperature enzyme-assisted cell wall disruption: Take 400 g of fresh onion bulbs, wash and drain them, then chop them. Add extraction buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.18% cellulase, 0.09% pectinase and 0.045% hemicellulase) at a ratio of 1:2, and place it in a 4°C environment, stirring at 75 rpm for 11 hours. After stirring, use a tissue homogenizer to homogenize at 7,500 rpm for 4 minutes to obtain onion tissue homogenate.

[0064] (2) Primary clarification: The obtained onion tissue homogenate was transferred to a centrifuge tube and centrifuged at 4°C and 4,200 g for 30 minutes. The supernatant was collected to obtain a preliminarily clarified onion homogenate of about 1,200 mL.

[0065] (3) Microfiltration pretreatment: The clarified onion homogenate was microfiltered through a 5 μm polyethersulfone membrane at an operating temperature of 5.5°C and a membrane flux of 38 L / m² / h. Approximately 1,150 mL of filtrate was collected for later use.

[0066] (4) Tangential flow ultrafiltration: The microfiltration filtrate was ultrafiltered through a polyethersulfone hollow fiber tangential flow filtration system with a molecular weight cutoff of 500 kDa. The ultrafiltration operating temperature was 5.5°C, the transmembrane pressure difference was 0.14 bar, and the tangential flow rate was 130 cm / s. Constant volume ultrafiltration was performed to concentrate the solution to 1 / 7 of its original volume (approximately 165 mL). Subsequently, the solution was eluted by dialyzing with 5 volumes of 20 mM HEPES pH 7.4, 150 mM NaCl buffer (approximately 825 mL). Approximately 160 mL of the filtrate was collected to obtain a solution rich in exosomes.

[0067] (5) Tangential flow nanofiltration: The ultrafiltration filtrate was subjected to nanofiltration through a polyethersulfone hollow fiber tangential flow filtration system with a molecular weight cutoff of 100 kDa. The nanofiltration operating temperature was 5.5°C, the transmembrane pressure difference was 0.09 bar, and the tangential flow rate was 95 cm / s. The solution was concentrated to 1 / 16 of its original volume (approximately 10 mL). The retentate was collected to obtain a concentrated onion exosome solution.

[0068] (6) Density gradient separation: Iodixanol and sucrose mixed density gradient solutions were prepared using the same method as in Example 1, and density gradient separation was performed. Exosome layer with a density of approximately 1.16 g / mL, approximately 1.4 mL, was collected.

[0069] (7) Buffer replacement: Buffer replacement was performed using the same method as in Example 1 to obtain approximately 1.1 mL of purified onion exosomes.

[0070] (8) Addition of cryoprotectant: The concentration of the obtained onion exosomes was determined to be 2.7 mg / mL. The cryoprotectant was added according to the following mass fraction ratios: 2.2% sodium carboxymethyl cellulose, 2.8% sodium alginate, 4.2% polyvinylpyrrolidone K30, and 0.8% hydroxypropyl-β-cyclodextrin. Specifically, 24.2 mg of sodium carboxymethyl cellulose, 30.8 mg of sodium alginate, 46.2 mg of polyvinylpyrrolidone K30, and 8.8 mg of hydroxypropyl-β-cyclodextrin were added to 1.1 mL of onion exosome solution. The mixture was gently stirred until the protectant was completely dissolved, yielding a liquid formulation of the onion exosome complex.

[0071] (9) Preparation of dry powder: freeze-drying was carried out in the same way as in Example 1 to obtain onion exosome complex dry powder.

[0072] Example 5: Preparation of rose exosome complex

[0073] This embodiment provides a method for preparing a rose exosome complex based on ultrapure technology, specifically including the following steps:

[0074] (1) Low-temperature enzyme-assisted cell wall disruption: Take 250 g of fresh rose petals, wash and drain them, then chop them. Add extraction buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.22% cellulase, 0.11% pectinase and 0.055% hemicellulase) at a ratio of 1:2, and place it in a 4°C environment, stirring at 85 rpm for 10 hours. After stirring, use a tissue homogenizer to homogenize at 8,500 rpm for 3.5 minutes to obtain rose tissue homogenate.

[0075] (2) Primary clarification: The obtained rose tissue homogenate was transferred to a centrifuge tube and centrifuged at 3,800 g for 30 minutes at 4°C. The supernatant was collected to obtain approximately 750 mL of preliminarily clarified rose homogenate.

[0076] (3) Microfiltration pretreatment: The clarified rose homogenate was microfiltered through a 5 μm polyethersulfone membrane at an operating temperature of 6.5°C and a membrane flux of 42 L / m² / h. Approximately 720 mL of filtrate was collected for later use.

[0077] (4) Tangential flow ultrafiltration: The microfiltration filtrate was ultrafiltered through a polyethersulfone hollow fiber tangential flow filtration system with a molecular weight cutoff of 500 kDa. The ultrafiltration operating temperature was 6.5°C, the transmembrane pressure difference was 0.16 bar, and the tangential flow rate was 125 cm / s. Constant volume ultrafiltration was performed to concentrate the solution to 1 / 9 of its original volume (approximately 80 mL). Subsequently, the solution was eluted by dialysis with 5 volumes of 20 mM HEPES pH 7.4, 150 mM NaCl buffer (approximately 400 mL). Approximately 75 mL of the filtrate was collected to obtain a solution rich in exosomes.

[0078] (5) Tangential flow nanofiltration: The ultrafiltration filtrate was subjected to nanofiltration through a polyethersulfone hollow fiber tangential flow filtration system with a molecular weight cutoff of 100 kDa. The nanofiltration operating temperature was 6.5°C, the transmembrane pressure difference was 0.11 bar, and the tangential flow rate was 105 cm / s. The solution was concentrated to 1 / 15 of its original volume (approximately 5 mL). The retentate was collected to obtain a concentrated rose exosome solution.

[0079] (6) Density gradient separation: Iodixanol and sucrose mixed density gradient solutions were prepared using the same method as in Example 1, and density gradient separation was performed. The exosome layer with a density of approximately 1.15 g / mL was collected, approximately 0.9 mL.

[0080] (7) Buffer replacement: Buffer replacement was performed using the same method as in Example 1 to obtain approximately 0.7 mL of purified rose exosomes.

[0081] (8) Addition of cryoprotectant: The concentration of rose exosomes obtained was determined to be 2.3 mg / mL. Cryoprotectant was added according to the following mass fraction ratios: 2.8% sodium carboxymethyl cellulose, 2.2% sodium alginate, 3.8% polyvinylpyrrolidone K30, and 1.2% hydroxypropyl-β-cyclodextrin. Specifically, 19.6 mg of sodium carboxymethyl cellulose, 15.4 mg of sodium alginate, 26.6 mg of polyvinylpyrrolidone K30, and 8.4 mg of hydroxypropyl-β-cyclodextrin were added to 0.7 mL of rose exosome solution, and the mixture was gently stirred until the protectant was completely dissolved, yielding a liquid formulation of the rose exosome complex.

[0082] (9) Preparation of dry powder: freeze-drying was carried out in the same way as in Example 1 to obtain dry powder of rose exosome complex.

[0083] Example 6: Preparation of Centella Asiatica exosome complex

[0084] This embodiment provides a method for preparing Centella asiatica exosome complex based on ultrapure technology, specifically including the following steps:

[0085] (1) Low-temperature enzyme-assisted cell wall disruption: Take 350 g of fresh whole Centella asiatica plant, wash and drain, then chop. Add extraction buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.2% cellulase, 0.1% pectinase and 0.05% hemicellulase) at a ratio of 1:2, place in a 4°C environment, and stir at 80 rpm for 10 hours. After stirring, use a tissue homogenizer to homogenize at 8,000 rpm for 4 minutes to obtain Centella asiatica tissue homogenate.

[0086] (2) Primary clarification: The obtained Centella asiatica tissue homogenate was transferred to a centrifuge tube and centrifuged at 4°C with a centrifugal force of 4,000g for 30 minutes. The supernatant was collected to obtain a preliminarily clarified Centella asiatica homogenate of about 1,050 mL.

[0087] (3) Microfiltration pretreatment: The clarified Centella asiatica homogenate was microfiltered through a 5 μm polyethersulfone membrane at an operating temperature of 6°C and a membrane flux of 40 L / m² / h. Approximately 1,000 mL of filtrate was collected for later use.

[0088] (4) Tangential flow ultrafiltration: The microfiltration filtrate was ultrafiltered through a polyethersulfone hollow fiber tangential flow filtration system with a molecular weight cutoff of 500 kDa. The ultrafiltration operating temperature was 6°C, the transmembrane pressure difference was 0.15 bar, and the tangential flow rate was 120 cm / s. Constant volume ultrafiltration was performed to concentrate the solution to 1 / 8 of its original volume (approximately 125 mL). Subsequently, the solution was eluted by dialyzing with 5 volumes of 20 mM HEPES pH 7.4, 150 mM NaCl buffer (approximately 625 mL). Approximately 120 mL of the filtrate was collected to obtain a solution rich in exosomes.

[0089] (5) Tangential flow nanofiltration: The ultrafiltration filtrate was subjected to nanofiltration through a polyethersulfone hollow fiber tangential flow filtration system with a molecular weight cutoff of 100 kDa. The nanofiltration operating temperature was 6°C, the transmembrane pressure difference was 0.1 bar, and the tangential flow rate was 100 cm / s. The solution was concentrated to 1 / 15 of its original volume (approximately 8 mL). The retentate was collected to obtain a concentrated Centella asiatica exosome solution.

[0090] (6) Density gradient separation: Iodixanol and sucrose mixed density gradient solutions were prepared using the same method as in Example 1, and density gradient separation was performed. Exosome layer with a density of approximately 1.18 g / mL, approximately 1.2 mL, was collected.

[0091] (7) Buffer replacement: Buffer replacement was performed using the same method as in Example 1 to obtain approximately 1.0 mL of purified Centella Asiatica exosomes.

[0092] (8) Addition of cryoprotectant: The concentration of Centella asiatica exosomes obtained was determined to be 2.8 mg / mL. Cryoprotectant was added according to the following mass fraction ratios: 2% sodium carboxymethyl cellulose, 3% sodium alginate, 4% polyvinylpyrrolidone K30, and 1% hydroxypropyl-β-cyclodextrin. Specifically, 20 mg of sodium carboxymethyl cellulose, 30 mg of sodium alginate, 40 mg of polyvinylpyrrolidone K30, and 10 mg of hydroxypropyl-β-cyclodextrin were added to 1.0 mL of Centella asiatica exosome solution, and the mixture was gently stirred until the protectant was completely dissolved, yielding a liquid preparation of the Centella asiatica exosome complex.

[0093] (9) Preparation of dry powder: freeze-drying was carried out in the same way as in Example 1 to obtain dry powder of Centella asiatica exosome complex.

[0094] Comparative Example 1: Citrus exosomes prepared by conventional ultracentrifugation

[0095] This comparative example uses the traditional ultracentrifugation method to prepare citrus exosomes. The specific steps are as follows:

[0096] (1) Take 500 g of fresh citrus peel, wash and drain it, then chop it and add 1,000 mL of PBS buffer (pH 7.4).

[0097] (2) Use a tissue homogenizer to homogenize at 8,000 rpm for 5 minutes to obtain citrus tissue homogenate;

[0098] (3) Centrifuge at 300 g for 10 minutes at 4°C to remove large tissue fragments;

[0099] (4) Collect the supernatant and centrifuge at 2,000 g for 20 minutes at 4°C to remove cell debris;

[0100] (5) Collect the supernatant and centrifuge at 10,000 g for 30 minutes at 4°C to remove organelles;

[0101] (6) Collect the supernatant and centrifuge at 100,000 g for 70 minutes at 4°C to collect the precipitate (exosomes).

[0102] (7) Resuspend the precipitate in PBS buffer and centrifuge again at 100,000 g for 70 minutes at 4°C, and collect the precipitate;

[0103] (8) Resuspend the precipitate in a small amount of PBS buffer to obtain citrus exosomes.

[0104] Comparative Example 2: Citrus exosomes without added cryo-drying protectant

[0105] This comparative example uses the same method as Example 1 to prepare citrus exosomes, but in the last step, no low-temperature freeze-drying protectant is added. The purified citrus exosome solution is directly dispensed into freeze-drying bottles, pre-frozen at -80°C for 2 hours, and then freeze-dried at -55°C for 48 hours to obtain citrus exosome powder.

[0106] Comparative Example 3: Green tea exosomes separated using a sucrose density gradient

[0107] This comparative example uses a method similar to that in Example 2 to prepare green tea exosomes, but in the density gradient separation step, a pure sucrose density gradient is used instead of the iodixanol / sucrose mixed density gradient. Specifically, a sucrose density gradient solution is prepared, divided into 5 layers: layer 1 is 10% sucrose, layer 2 is 20% sucrose, layer 3 is 30% sucrose, layer 4 is 40% sucrose, and layer 5 is 50% sucrose. Each layer of the density gradient solution is carefully added sequentially from bottom to top into an ultracentrifuge tube, 2 mL per layer. Finally, the concentrated green tea exosome solution is carefully added to the top layer of the density gradient. The mixture is centrifuged at 100,000 g for 16 hours at 4°C. After centrifugation, the interfaces are carefully separated from top to bottom, and the exosome layer with a density of approximately 1.16-1.18 g / mL (located between the 30% and 40% sucrose layers) is collected.

[0108] Comparative Example 4: Grape exosomes without enzyme-assisted cell wall disruption

[0109] This comparative example uses a similar method to Example 3 to prepare grape exosomes, but instead of enzyme-assisted cell disruption, mechanical homogenization is used directly in the first step. Specifically, 600 g of fresh grapes are taken, washed, drained, and seeded. 1,200 mL of extraction buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EDTA, no enzyme added) is added, and the mixture is homogenized directly using a tissue homogenizer at 9,000 rpm for 5 minutes to obtain grape tissue homogenate. Subsequent steps are the same as in Example 3.

[0110] Comparative Example 5: Onion exosomes without tangential flow filtration

[0111] This comparative example uses a method similar to that in Example 4 to prepare onion exosomes, but instead of tangential flow filtration, it employs multiple differential centrifugations. Specifically, 400 g of fresh onion bulbs are subjected to enzyme-assisted cell disruption and primary clarification as described in Example 4. After obtaining the clarified liquid, it is centrifuged at 10,000 g for 30 minutes at 4°C to remove organelles and macromolecular impurities. The supernatant is collected and centrifuged at 100,000 g for 70 minutes at 4°C to collect the precipitate. The precipitate is resuspended in 20 mM HEPES pH 7.4, 150 mM NaCl buffer. Subsequent steps are the same as density gradient separation and subsequent steps in Example 4.

[0112] Comparative Example 6: Rose exosomes treated with a single protectant

[0113] This comparative example uses the same method as Example 5 to prepare rose exosomes, but when adding the cryoprotectant, only polyvinylpyrrolidone K30 is used as the protectant, and the amount added is 10%. Specifically, 70 mg of polyvinylpyrrolidone K30 is added to 0.7 mL of rose exosome solution, and the mixture is gently stirred to completely dissolve the protectant, resulting in a liquid formulation of the rose exosome complex. The subsequent lyophilization steps are the same as in Example 5.

[0114] Comparative Example 7: Centella Asiatica exosomes using traditional solid-film components

[0115] This comparative example uses the same method as Example 6 to prepare Centella asiatica exosomes, but when adding the cryoprotectant, it uses the traditional solid-film components, namely 5% mannitol, 1.5% trehalose, and 0.8% maltodextrin. Specifically, 50 mg of mannitol, 15 mg of trehalose, and 8 mg of maltodextrin are added to 1.0 mL of Centella asiatica exosome solution, and the mixture is gently stirred to completely dissolve the protectant, yielding a liquid formulation of the Centella asiatica exosome complex. Subsequent freeze-drying steps are the same as in Example 6.

[0116] Experimental Example 1: Plant Exosome Particle Size Analysis

[0117] The particle size of plant exosomes prepared in each example and comparative example was analyzed using dynamic light scattering (DLS). A Malvern Zetasizer Nano ZS90 nanoparticle size analyzer was used, and the test temperature was 25°C. Each sample was tested three times, and the average value was taken. The results are shown in Table 1.

[0118] Table 1. Particle size characteristics of plant exosomes in each example and comparative example.

[0119] Sample number Average particle size (nm) Particle size distribution range (nm) Polydispersion Index (PDI) Example 1 95 50-150 0.132 Example 2 75 40-120 0.158 Example 3 110 60-180 0.145 Example 4 85 45-140 0.138 Example 5 90 50-160 0.152 Example 6 60 30-100 0.127 Comparative Example 1 125 50-320 0.386 Comparative Example 3 80 40-130 0.275 Comparative Example 4 130 60-250 0.322 Comparative Example 5 115 45-310 0.358

[0120] As can be seen from the results in Table 1, the plant exosomes prepared in the embodiments of the present invention have a narrow particle size distribution range and a small polydispersity coefficient (both less than 0.2), indicating good particle size uniformity. In contrast, the plant exosomes prepared in the comparative examples have a wide particle size distribution range and a large polydispersity coefficient (both greater than 0.25), indicating poor particle size uniformity. In particular, the polydispersity coefficients of Comparative Example 1, prepared using the traditional ultracentrifugation method, and Comparative Example 5, which did not use tangential flow filtration technology, are as high as 0.386 and 0.358, respectively, indicating that these methods are difficult to obtain exosomes with uniform particle size.

[0121] Experimental Example 2: Analysis of Plant Exosome Yield and Purity

[0122] The yield and purity of plant exosomes prepared in each example and comparative example were analyzed. Yield was expressed as the amount of exosome protein obtained per gram of fresh plant weight (μg / g) and was determined using a BCA protein quantification kit. Purity was assessed by analyzing the relative content ratios of exosome marker proteins (CD63, TSG101, and Alix) to impurity proteins (cytochrome C and calreticulin) using a microfluidic electrophoresis system (Agilent 2100 Bioanalyzer).

[0123] Experimental Methods: 10 μL of exosome sample was added to 90 μL of lysis buffer (containing 1% SDS and protease inhibitor), and lysed on ice for 30 minutes. Insoluble matter was removed by centrifugation at 12,000 g for 15 minutes, and the supernatant was collected. Protein concentration was determined using a BCA protein quantification kit (Thermo Scientific Pierce). A suitable amount of protein sample was analyzed by microfluidic electrophoresis, and the relative content ratio of exosome marker proteins to impurity proteins was calculated. Results are shown in Table 2.

[0124] Table 2. Yield and purity of plant exosomes in each example and comparative example.

[0125] Sample number Yield (μg / g) Exosome marker to impurity protein ratio Relative purity (%) Example 1 15.3 8.6 100 Example 2 12.7 8.2 95 Example 3 18.5 8.9 103 Example 4 14.2 8.4 98 Example 5 13.5 8.3 97 Example 6 16.8 8.7 101 Comparative Example 1 8.2 4.1 48 Comparative Example 3 10.5 6.2 72 Comparative Example 4 7.3 3.8 44 Comparative Example 5 9.1 4.5 52 Comparative Example 7 16.2 8.5 99

[0126] As can be seen from the results in Table 2, the yield of plant exosomes prepared in the embodiments of the present invention is significantly higher than that of Comparative Examples 1, 3, 4, and 5. The ratio of exosome markers to impurity proteins is also significantly higher than in these comparative examples, indicating that the method of the present invention not only improves the extraction efficiency of exosomes but also significantly improves the purity of exosomes. It is worth noting that although the yield and purity of Comparative Example 7 are close to those of the embodiments of the present invention, as shown in subsequent experiments, its stability is significantly insufficient.

[0127] Experimental Example 3: Stability Analysis of Plant Exosome Complexes

[0128] Stability analysis was performed on the plant exosome complexes prepared in each example and Comparative Examples 2, 6, and 7, including the stability of the liquid formulation at 4°C and the stability of the lyophilized formulation at 25°C. Stability was assessed by changes in exosome particle size, protein content, and the content of active ingredients (polyphenols and flavonoids).

[0129] Experimental methods:

[0130] (1) Stability test of liquid formulations: The liquid formulations of each sample were stored at 4°C for 6 months, and samples were taken monthly to determine the particle size, protein content and active ingredient content. The particle size was determined by dynamic light scattering method; the protein content was determined by BCA protein quantification kit; the active ingredient content was determined by Folin-Ciocalteu method for total polyphenol content and AlCl3 colorimetric method for total flavonoid content.

[0131] (2) Stability test of lyophilized formulations: The lyophilized formulations of each sample were stored at 25°C for 18 months. Samples were taken every 3 months, rehydrated with an equal volume of ultrapure water, and the particle size, protein content and active ingredient content were determined. The determination method was the same as above.

[0132] The results are shown in Tables 3 and 4:

[0133] Table 3. Stability of liquid formulations after 6 months of storage at 4°C

[0134] Sample number Particle size change rate (%) Protein content change rate (%) Change rate of active ingredient content (%) Example 1 4.2 3.5 5.1 Example 2 5.1 4.2 6.3 Example 3 3.8 3 4.5 Example 4 4.5 3.8 5.7 Example 5 4.8 4 5.9 Example 6 4 3.2 4.8 Comparative Example 2 32.5 28.7 45.2 Comparative Example 6 18.7 15.3 22.6 Comparative Example 7 12.3 10.5 15.8

[0135] Table 4. Stability of lyophilized formulations after 18 months of storage at 25°C

[0136] Sample number Particle size change rate after rehydration (%) Protein content change rate (%) Change rate of active ingredient content (%) Example 1 6.5 5.2 7.8 Example 2 7.3 6 9.1 Example 3 5.7 4.5 6.9 Example 4 6.8 5.5 8.4 Example 5 7 5.8 8.7 Example 6 6.2 4.9 7.5 Comparative Example 2 58.3 52.1 73.6 Comparative Example 6 35.2 28.6 41.5 Comparative Example 7 23.6 18.9 27.3

[0137] As can be seen from the results in Tables 3 and 4, the plant exosome complexes prepared in the embodiments of the present invention exhibit excellent stability in both liquid and lyophilized states, with minimal changes in particle size, protein content, and active ingredient content (less than 7% change rate after 6 months of storage for the liquid formulation, and less than 10% change rate after 18 months of storage for the lyophilized formulation). In contrast, Comparative Example 2 (without a cryoprotectant), Comparative Example 6 (using a single protectant), and Comparative Example 7 (using a traditional solid-film component) showed varying degrees of particle size increase, protein content decrease, and active ingredient content reduction during storage, indicating that the exosome structure and active ingredients underwent varying degrees of damage. Among these, Comparative Example 2 (without any protectant) exhibited the worst stability; after 18 months of storage, the protein content of the lyophilized formulation decreased by more than 50%, and the active ingredient content decreased by more than 70%.

[0138] Experimental Example 4: Bioactivity Analysis of Plant Exosome Complexes

[0139] The plant exosome complexes prepared in Examples 1, 3, 1, 4, and 7 were subjected to bioactivity analysis, including antioxidant activity, cell protection, and transdermal permeability.

[0140] Experimental methods:

[0141] (1) Antioxidant activity test: The antioxidant activity was determined by the DPPH free radical scavenging ability method. 100 μL of exosome complex samples of different concentrations (protein concentration of 0.1-1.0 mg / mL) were added to a 96-well plate, and 100 μL of 0.1 mM DPPH methanol solution was added. After reacting at room temperature in the dark for 30 minutes, the absorbance was measured at a wavelength of 517 nm and the DPPH free radical scavenging rate was calculated.

[0142] (2) Cell protection test: The cell protection effect was evaluated using a H2O2-induced human epidermal keratinocyte (HaCaT) injury model. HaCaT cells were seeded at a density of 5 × 10^4 cells / well in 96-well plates and cultured for 24 hours. Then, different concentrations of exosome complex samples (protein concentration of 0.01-0.1 mg / mL) were added for pretreatment for 2 hours, followed by treatment with 500 μM H2O2 for 1 hour. The cells were washed with PBS, cultured in medium containing 10% CCK-8 reagent for another 4 hours, and the absorbance was measured at 450 nm to calculate cell viability.

[0143] (3) Transdermal permeability test: Transdermal permeability was determined using the Franz diffusion cell model. Pigskin (approximately 500 μm thick) was fixed between the upper and lower parts of the Franz diffusion cell. PBS solution containing 1 mg / mL of exosome complex sample (containing 0.1% fluorescein-labeled exosomes for tracing) was added to the upper part, and PBS receiving solution was added to the lower part. The mixture was stirred at 37°C, and samples were taken from the receiving solution periodically. Fluorescence intensity was measured using a fluorescence spectrophotometer (excitation wavelength 485 nm, emission wavelength 520 nm), and the cumulative permeation was calculated.

[0144] The results are shown in Table 5:

[0145] Table 5. Bioactivity of plant exosome complexes

[0146] Sample number DPPH free radical scavenging rate (%) Cell viability (%) Cumulative permeability (μg / cm²·h) Example 1 78.5 85.3 12.7 Example 3 82.3 88.1 13.5 Comparative Example 1 42.1 56.7 5.3 Comparative Example 4 38.5 52.3 4.8 Comparative Example 7 68.2 74.5 10.2

[0147] As shown in Table 5, the plant exosome complexes prepared in the embodiments of the present invention exhibit significant antioxidant activity (DPPH free radical scavenging rate greater than 75%) and cell protection effects (H2O2-induced cell viability greater than 85%), while also demonstrating good transdermal permeability (cumulative permeation greater than 12 μg / cm²·h). In contrast, the bioactivity of Comparative Example 1, prepared using the conventional ultracentrifugation method, and Comparative Example 4, prepared without enzyme-assisted cell disruption, was significantly lower, indicating that the method of the present invention not only improves the yield and purity of exosomes but also maintains their bioactivity. Although Comparative Example 7, using a traditional solid membrane component, also showed some bioactivity, it was still significantly lower than that of the embodiments of the present invention, further demonstrating the superiority of the low-temperature freeze-drying protectant formulation used in the present invention.

[0148] Experimental Example 5: Analysis of Active Components in Plant Exosome Complexes

[0149] The plant exosome complexes prepared in Examples 1-6 and Comparative Examples 1, 4, and 7 were analyzed for their active ingredients, including total polyphenol content, total flavonoid content, and bioactive protein content.

[0150] Experimental methods:

[0151] (1) Determination of total polyphenol content: The Folin-Ciocalteu method was used. 100 μL of exosome complex sample (protein concentration adjusted to 1.0 mg / mL) was added to a 96-well plate, along with 10 μL of Folin-Ciocalteu reagent and 90 μL of 10% Na2CO3 solution. After reacting at room temperature in the dark for 30 minutes, the absorbance was measured at 765 nm. A standard curve was plotted using gallic acid as a standard, and the total polyphenol content (expressed as gallic acid equivalent, mg GAE / g protein) was calculated.

[0152] (2) Determination of total flavonoid content: The AlCl3 colorimetric method was used. 100 μL of exosome complex sample (protein concentration adjusted to 1.0 mg / mL) was added to a 96-well plate, along with 10 μL of 10% AlCl3 solution and 90 μL of 1 M NaOH solution. After reacting at room temperature in the dark for 10 minutes, the absorbance was measured at 510 nm. A standard curve was plotted using rutin as a standard, and the total flavonoid content (expressed as rutin equivalent, mg RE / g protein) was calculated.

[0153] (3) Determination of bioactive protein content: The content of bioactive proteins such as SOD1, GPX1 and HSP70 in exosomes was determined by enzyme-linked immunosorbent assay (ELISA). The corresponding ELISA kits were used, and the operation was performed according to the kit instructions to calculate the content of each bioactive protein (μg / g protein).

[0154] The results are shown in Table 6:

[0155] Table 6. Content of active ingredients in plant exosome complexes

[0156] Sample number Total polyphenol content (mg GAE / g protein) Total flavonoid content (mgRE / g protein) SOD1 content (μg / g protein) GPX1 content (μg / g protein) HSP70 content (μg / g protein) Example 1 42.5 18.3 456 312 625 Example 2 65.8 28.7 523 345 682 Example 3 38.2 15.6 412 287 598 Example 4 31.5 12.8 378 265 562 Example 5 52.3 23.1 485 328 647 Example 6 47.6 20.5 468 319 635 Comparative Example 1 21.2 8.5 225 156 315 Comparative Example 4 18.7 7.2 198 142 287 Comparative Example 7 36.8 15.1 398 275 578

[0157] As can be seen from the results in Table 6, the plant exosome complexes prepared in the embodiments of the present invention contain abundant active ingredients, including polyphenolic compounds, flavonoids, and bioactive proteins such as SOD1, GPX1, and HSP70. In contrast, the content of active ingredients in Comparative Examples 1 and 4 is significantly lower, only 40-50% of that in the embodiments of the present invention. Although the content of active ingredients in Comparative Example 7 is slightly higher than that in Comparative Examples 1 and 4, it is still significantly lower than that in the embodiments of the present invention, indicating that the preparation method of the present invention can better preserve the active ingredients in plant exosomes.

[0158] Experimental Example 6: Performance Evaluation of Plant Exosome Complex

[0159] To evaluate the performance of the plant exosome complexes prepared in this invention in practical applications, Examples 1 (citrus exosome complex), 2 (green tea exosome complex), and 5 (rose exosome complex) were selected for skincare efficacy evaluation, including skin moisturizing, anti-aging, and repair properties. Comparative Examples 1, 3, and 7 were used as control groups.

[0160] Experimental methods:

[0161] (1) Skin moisturizing assessment: The in vitro stratum corneum water content determination method was used. Human stratum corneum samples were immersed in a solution containing 0.5% exosome complex for 30 minutes, and then equilibrated at room temperature (25°C, 50% relative humidity) for 1 hour. The stratum corneum water content was measured using a skin moisture meter (Corneometer CM825). Each sample was measured 5 times, and the average value was taken.

[0162] (2) Anti-aging assessment: Human skin fibroblasts (HSFC) model was used. HSFCs were seeded at a density of 1×10^4 cells / well in 96-well plates and cultured for 24 hours. Then, culture medium containing 0.1% exosome complex was added for 48 hours. The expression levels of anti-aging related genes (such as COL1A1, ELN, and TIMP1) were determined by real-time quantitative PCR. β-actin was used as an internal reference gene, and the relative expression level was calculated using the 2^(-ΔΔCt) method.

[0163] (3) Repair assessment: A human epidermal keratinocyte (HaCaT) scratch model was used. HaCaT cells were seeded at a density of 5 × 10^5 cells / well in 24-well plates and cultured until the cell monolayers reached confluence. A vertical "wound" was then made on the cell layer using a sterile 200 μL pipette tip. After washing with PBS, serum-free medium containing 0.2% exosome complex was added for treatment. The healing status of the "wound" was photographed and recorded at 0, 12, 24, and 48 hours after the scratch. The "wound" area was measured using ImageJ software, and the healing rate was calculated.

[0164] The results are shown in Tables 7, 8, and 9:

[0165] Table 7. Results of skin moisturizing effect assessment of plant exosome complex.

[0166] Sample number stratum corneum moisture content (au) Relative moisturizing effect (%) Blank control 32.5 ± 2.8 0 Example 1 58.7 ± 4.3 80.6 Example 2 62.5 ± 5.1 92.3 Example 5 65.3 ± 4.8 100.9 Comparative Example 1 42.3 ± 3.5 30.2 Comparative Example 3 48.6 ± 4.2 49.5 Comparative Example 7 54.1 ± 4.5 66.5 Hyaluronic acid (0.5%) 64.8 ± 5.0 99.4

[0167] Table 8. Evaluation results of the anti-aging properties of plant exosome complexes (relative gene expression levels)

[0168] Sample number COL1A1 ELN TIMP1 Average anti-aging effect (%) Blank control 1 1 1 0 Example 1 2.35 1.85 2.12 111.7 Example 2 2.58 2.03 2.32 131 Example 5 2.42 1.95 2.23 120 Comparative Example 1 1.45 1.32 1.4 39 Comparative Example 3 1.73 1.58 1.65 65.3 Comparative Example 7 2.08 1.72 1.95 91.7 Retinol (0.05%) 2.48 1.98 2.25 123.7

[0169] Table 9. Results of the reparative assessment of plant exosome complexes (scratch healing rate after 48 hours)

[0170] Sample number Healing rate (%) Relative repair effect (%) Blank control 52.5 ± 4.2 0 Example 1 85.3 ± 6.8 85.3 Example 2 83.7 ± 7.2 81.4 Example 5 88.5 ± 7.5 93.3 Comparative Example 1 65.8 ± 5.3 34.6 Comparative Example 3 72.4 ± 6.1 51.8 Comparative Example 7 79.6 ± 6.5 70.5 EGF (10 ng / mL) 89.2 ± 7.8 95.2

[0171] As can be seen from the results in Tables 7-9, the plant exosome complexes prepared in the embodiments of the present invention exhibit excellent performance in skin moisturizing, anti-aging, and repair properties, approaching or reaching the levels of commercially available skincare active ingredients (such as hyaluronic acid, retinol, and EGF). Among them, Example 5 (rose exosome complex) showed the most outstanding performance in skin moisturizing and repair properties, while Example 2 (green tea exosome complex) showed the best performance in anti-aging properties. In contrast, the skincare efficacy of Comparative Examples 1, 3, and 7 was significantly lower than that of the embodiments of the present invention, indicating that the preparation method of the present invention can better maintain the bioactivity and efficacy of plant exosomes.

[0172] This invention innovatively combines tangential flow filtration technology with iodixanol / sucrose density gradient separation technology, and adds a specific low-temperature freeze-drying protectant, successfully solving the technical problems of low purity, uneven particle size, and unstable activity of plant exosomes in existing technologies. The working mechanism and technical advantages of this invention are mainly reflected in the following aspects:

[0173] 1. Optimized Mechanism of Low-Temperature Enzyme-Assisted Cell Wall Disruption: This invention employs low-temperature (4°C) conditions for enzyme-assisted cell wall disruption, instead of the traditional room temperature or high-temperature conditions. The key mechanism of this improvement lies in the fact that low temperature significantly reduces the activity of endogenous proteases in plant tissues, thereby reducing the degradation of exosome membrane proteins. Simultaneously, low temperature also reduces the activity of oxidases such as polyphenol oxidase in plant tissues, reducing the oxidation and polymerization of polyphenols and preventing these polymers from non-specifically binding to exosomes, thus affecting the purity and activity of exosomes. Furthermore, this invention precisely controls the ratio of cellulase, pectinase, and hemicellulase. The synergistic effect of these three enzymes can efficiently degrade the three major components of plant cell walls (cellulose, pectin, and hemicellulose), maximizing the release of exosomes while minimizing damage to the exosome structure.

[0174] The results of Experiments 4 and 5 demonstrate that, compared to Comparative Example 4 which did not employ enzyme-assisted cell disruption, the plant exosomes prepared in the embodiments of the present invention maintained higher biological activity and higher content of active ingredients. This is because enzyme-assisted cell disruption technology can efficiently release exosomes from plant cells under mild conditions, avoiding the structural damage and loss of active ingredients that may result from traditional mechanical disruption methods.

[0175] 2. Optimization Mechanism of Tangential Flow Filtration Technology: This invention replaces traditional multiple differential centrifugation with tangential flow filtration technology. The key mechanism of this improvement lies in the fact that in tangential flow filtration, the sample liquid flows parallel to the membrane surface, and the resulting tangential flow continuously cleans the membrane surface, preventing exosomes from accumulating and aggregating on the membrane surface, and significantly reducing shear damage to exosomes. Simultaneously, by precisely controlling the operating parameters of ultrafiltration and nanofiltration (such as temperature, transmembrane pressure difference, flow rate, etc.), gentle yet efficient separation and concentration of exosomes are achieved.

[0176] In the tangential flow ultrafiltration step, this invention uses a membrane with a molecular weight cutoff of 500 kDa, which can effectively remove small molecule impurities and most proteins, while retaining exosomes to the maximum extent. In the tangential flow nanofiltration step, this invention uses a membrane with a molecular weight cutoff of 100 kDa, which can further concentrate exosomes and improve the efficiency of subsequent density gradient separation.

[0177] The results of Experiments 2 and 3 show that, compared with Comparative Example 1 (using conventional ultracentrifugation) and Comparative Example 5 (not using tangential flow filtration), the plant exosomes prepared in the embodiments of the present invention have higher yield, purity, and stability. This fully demonstrates the superiority of tangential flow filtration technology in the isolation and purification of plant exosomes.

[0178] 3. Optimization Mechanism of Iodixanol / Sucrose Dual-Gradient Separation: This invention employs a dual-gradient density system formed by mixing iodixanol and sucrose, instead of the traditional single sucrose density gradient. The key mechanism of this improvement lies in the fact that iodixanol, as a non-ionic isotonic contrast agent, has the characteristics of low osmotic pressure and low viscosity, which can better maintain the original morphology of exosomes during ultracentrifugation and reduce exosome deformation and aggregation. At the same time, the dual-gradient system formed by sucrose and iodixanol provides a finer density distribution, which can achieve more accurate separation based on the density differences of plant exosomes, resulting in exosomes with higher purity and particle size uniformity.

[0179] This invention establishes a gradient system consisting of five layers of different densities by optimizing the ratio of iodixanol to sucrose (layer 1: 5% iodixanol and 5% sucrose; layer 2: 10% iodixanol and 10% sucrose; layer 3: 20% iodixanol and 15% sucrose; layer 4: 30% iodixanol and 20% sucrose; layer 5: 40% iodixanol and 25% sucrose). This gradient design can provide finer resolution within a density range of 1.15-1.19 g / mL (i.e., the density range of most plant exosomes), thereby achieving efficient separation of exosomes.

[0180] The results of Experiments 1 and 2 show that, compared with Comparative Example 3 which uses pure sucrose density gradient separation, the plant exosomes prepared in the embodiments of the present invention have better particle size uniformity and higher purity, which fully demonstrates the superiority of the iodixanol / sucrose dual gradient separation technology.

[0181] 4. Optimization Mechanism of Low-Temperature Lyophilization Protectant Formulation: This invention develops a low-temperature lyophilization protectant formulation composed of sodium carboxymethyl cellulose, sodium alginate, polyvinylpyrrolidone K30, and hydroxypropyl-β-cyclodextrin. The working mechanism of this innovative formulation is based on multiple synergistic protective effects:

[0182] (1) Sodium carboxymethyl cellulose, as an anionic polymer, has excellent film-forming and water-retaining properties. It can form a protective layer on the surface of exosomes to prevent ice crystal growth from damaging the exosome membrane structure during freeze-drying. At the same time, its abundant carboxyl and hydroxyl groups can interact with the polar groups on the surface of the exosome membrane through hydrogen bonds, thus stabilizing the exosome structure.

[0183] (2) Sodium alginate is a natural polysaccharide with good biocompatibility and gel-forming ability. It can form a three-dimensional network structure during freeze-drying, fix exosomes and reduce their aggregation. At the same time, the carboxyl and hydroxyl groups in sodium alginate can interact with the polar groups on the surface of the exosome membrane through hydrogen bonds, further stabilizing the exosome structure.

[0184] (3) Polyvinylpyrrolidone K30 (PVP K30) is a non-ionic water-soluble polymer with good film-forming properties and high glass transition temperature. It can form an amorphous glassy structure during freeze-drying, reducing collapse and shrinkage during freeze-drying. At the same time, the pyrrolidone groups of PVP K30 can interact with various groups on the surface of exosome membranes through hydrogen bonds and van der Waals forces, providing additional protection.

[0185] (4) Hydroxypropyl-β-cyclodextrin is a cyclic oligosaccharide with a unique barrel-shaped structure. It is hydrophobic inside and hydrophilic outside. It can interact with lipid molecules in the exosome membrane through its internal hydrophobic cavity to stabilize the lipid bilayer structure. At the same time, its multiple hydroxyl groups can interact with water molecules and other protective molecules through hydrogen bonds to form a more stable protective network.

[0186] The synergistic effect of these four components forms a multi-level, multi-mechanism protection system that can effectively maintain the structural integrity and biological activity of exosomes, enabling them to exhibit excellent stability in both liquid preservation and freeze-drying states.

[0187] The results of Experiment 3 show that, compared with Comparative Example 2 (without adding a low-temperature freeze-drying protectant), Comparative Example 6 (using a single protectant), and Comparative Example 7 (using a traditional solid film component), the plant exosome complex prepared in the embodiments of the present invention exhibits superior stability in both liquid and freeze-dried states, fully demonstrating the superiority of the low-temperature freeze-drying protectant formulation of the present invention.

[0188] Through the synergistic effect of the above multiple mechanisms, the present invention has the following significant advantages over the prior art:

[0189] (1) Improved the integrity and activity of plant exosomes: By using low-temperature enzyme-assisted cell disruption and tangential flow filtration technology, the shear damage to the exosome structure was significantly reduced, maintaining the integrity and biological activity of the exosomes. The results of Experiment Example 4 show that the plant exosome complex prepared in the embodiments of the present invention has significant antioxidant activity, cell protection effect and transdermal permeability, all of which are significantly better than the comparative example.

[0190] (2) Improved purity and uniformity of plant exosomes: High-precision separation of plant exosomes was achieved using the iodixanol / sucrose dual-gradient separation technique, resulting in exosomes with high purity and uniform particle size. The results of Experimental Examples 1 and 2 show that the plant exosomes prepared in the embodiments of the present invention have a narrow particle size distribution range, a small polydispersity coefficient, and high purity, all of which are significantly better than the comparative examples.

[0191] (3) Improved stability of plant exosome complexes: Through an innovative low-temperature freeze-drying protectant formulation, the structural integrity and biological activity of plant exosomes were effectively maintained, resulting in excellent stability under both liquid and freeze-dried conditions. The results of Experimental Example 3 show that the plant exosome complexes prepared in the embodiments of the present invention maintained high stability after 6 months of liquid storage and 18 months of freeze-dried storage, with a significantly lower rate of change than the comparative example.

[0192] (4) Improved application performance of plant exosome complex: The results of Experiment 6 show that the plant exosome complex prepared in the embodiments of the present invention exhibits excellent performance in skin moisturizing, anti-aging and repair, which is close to or reaches the level of commercial skin care efficacy ingredients, and is significantly better than the comparative example, providing technical support for the application of plant exosomes in the fields of beauty and skin care.

[0193] In summary, this invention, through the synergistic effect of multiple innovative technologies, successfully solves the technical problems of low purity, uneven particle size, and unstable activity of plant exosomes in the prior art, and prepares plant exosome complexes with high purity, high uniformity, high stability and high bioactivity, providing technical support for the application of plant exosomes in the fields of beauty, skin care, functional foods and drug carriers.

Claims

1. A method for preparing plant exosome complexes based on ultrapure technology, characterized in that, The preparation method includes the following steps: S1: Plant tissue homogenate was prepared by processing plant tissue using low-temperature enzyme-assisted cell disruption technology. S2: Perform primary clarification on the homogenate obtained in S1 to remove large particulate impurities; S3: The clarified liquid obtained in S2 is pretreated by microfiltration through a polyethersulfone membrane; S4: The filtrate obtained in S3 is subjected to tangential flow ultrafiltration to remove macromolecular impurities; S5: The filtrate obtained in S4 is enriched with exosomes by tangential flow nanofiltration; S6: The enriched solution obtained in S5 was subjected to density gradient separation by mixing iodixanol and sucrose, and exosomes in the corresponding density range were collected. S7: Replace the exosomes obtained in S6 with buffer solution to remove the density gradient agent; S8: Add a low-temperature freeze-drying protectant to the plant exosomes obtained in S7 to prepare a plant exosome complex.

2. The preparation method according to claim 1, characterized in that, The low-temperature enzyme-assisted cell disruption technology in S1 includes: Take fresh plant tissue, wash and drain it, then chop it. The plant material was mixed with the extraction buffer at a ratio of 1:

2. The extraction buffer consisted of 20 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EDTA, and 0.2% cellulase, 0.1% pectinase, and 0.05% hemicellulase. Stir at 60-120 rpm for 8-12 hours at 4°C; After stirring, homogenize the tissue in a homogenizer for 3-5 minutes to obtain a plant tissue homogenate.

3. The preparation method according to claim 1, characterized in that, The primary clarification method in S2 is as follows: The plant tissue homogenate obtained in S1 was centrifuged at 3,000-5,000g for 30 minutes at 4°C. Collect the supernatant to obtain a preliminarily clarified plant homogenate.

4. The preparation method according to claim 1, characterized in that, The microfiltration pretreatment method in S3 is as follows: The clarified liquid obtained from S2 was microfiltered through a 5μm polyethersulfone membrane, with the operating temperature controlled at 4-8°C and the membrane flux controlled at 30-50L / m² / h.

5. The preparation method according to claim 1, characterized in that, The tangential flow ultrafiltration method in S4 is as follows: The filtrate obtained in S3 was subjected to ultrafiltration through a polyethersulfone hollow fiber tangential flow filtration system with a molecular weight cutoff of 500 kDa. The ultrafiltration operating temperature is controlled at 4-8°C, the transmembrane pressure difference is controlled at 0.1-0.2 bar, and the tangential flow rate is controlled at 100-150 cm / s; Perform constant volume ultrafiltration to concentrate the volume to 1 / 5 to 1 / 10 of the original volume; A two-step dialysis elution method was used, with 5 volumes of 20 mM HEPES pH 7.4 and 150 mM NaCl buffer for dialysis elution. Collect the retentate from the filter membrane to obtain a solution rich in exosomes.

6. The preparation method according to claim 1, characterized in that, The tangential flow nanofiltration method in S5 is as follows: The filtrate obtained from S4 was subjected to nanofiltration through a polyethersulfone hollow fiber tangential flow filtration system with a molecular weight cutoff of 100 kDa. The nanofiltration operating temperature is controlled at 4-8°C, the transmembrane pressure difference is controlled at 0.05-0.15 bar, and the tangential flow rate is controlled at 80-120 cm / s; Concentrate the volume to 1 / 10-1 / 20 of the original volume; Collect the retentate from the filter membrane to obtain a concentrated exosome solution.

7. The preparation method according to claim 1, characterized in that, The density gradient separation method in S6 is as follows: Prepare a density gradient solution of iodixanol and sucrose, which consists of 5 layers: the first layer is 5% iodixanol and 5% sucrose, the second layer is 10% iodixanol and 10% sucrose, the third layer is 20% iodixanol and 15% sucrose, the fourth layer is 30% iodixanol and 20% sucrose, and the fifth layer is 40% iodixanol and 25% sucrose. Add the density gradient solutions of each layer to the ultracentrifuge tube from bottom to top, 2 mL for each layer; Finally, the concentrated exosome solution obtained in S5 was added to the top layer of the density gradient. Centrifuge at 100,000g for 16 hours at 4°C; Exosome layers with a density of approximately 1.15–1.19 g / mL were collected.

8. The preparation method according to claim 1, characterized in that, The buffer replacement method in S7 is as follows: The exosome layer obtained from S6 was replaced with buffer through an ultrafiltration tube with a molecular weight cutoff of 10 kDa. Add 10 volumes of 20 mM HEPES pH 7.4 and 150 mM NaCl buffer, and centrifuge at 3,000 g for 30 minutes at 4°C. Discard the filtrate and repeat the above steps three times to completely remove iodixanol and sucrose; Collect the retentate to obtain purified plant exosomes.

9. The preparation method according to claim 1, characterized in that, The cryogenic freeze-drying protectant in S8 is added according to the following mass fraction ratio: 2% sodium carboxymethyl cellulose, 3% sodium alginate, 4% polyvinylpyrrolidone K30, 1% hydroxypropyl-β-cyclodextrin.

10. The preparation method according to claim 1, characterized in that, The method for preparing the plant exosome complex dry powder in S8 is as follows: The plant exosome complex with added low-temperature freeze-drying protectant was dispensed into freeze-drying bottles; Pre-freeze at -80°C for 2 hours; The plant exosome complex was freeze-dried at -55°C for 48 hours to obtain a dry powder.