A high-antioxidant-activity seabuckthorn protoplast exosome reservation preparation method and application

Abstract

The application belongs to the technical field of biological preparation of plant-derived extracellular vesicles, and particularly relates to a preservation preparation method of seabuckthorn plasma exosomes with high antioxidant activity and application thereof. The application adopts a whole low-temperature pure physical coupling process, and is prepared through low-temperature gradient low-speed impurity removal, low-temperature low-pressure fractional ultrafiltration enrichment, isotonic buffer steady-state protection, and low-temperature vacuum freeze-drying shaping, thereby avoiding mechanical shearing, high-temperature oxidation and chemical reagent intervention in the whole process. The obtained exosomes have a particle size of 30-150 nm, a vesicle integrity preservation rate of greater than or equal to 92%, excellent DPPH, ABTS and superoxide anion scavenging capacity, and an overall antioxidant activity improved by more than 40% compared with traditional processes, and an activity attenuation rate of less than 8% after storage at room temperature for 6 months. The process parameters are controllable and suitable for large-scale production, there is no chemical residue, the safety is high, and the application can be applied to the fields of skin antioxidant repair preparations, functional health foods and oxidative stress damage repair drug adjuvants.

Description

Technical Field

[0001] This invention belongs to the field of plant-derived extracellular vesicle bioprocessing technology, specifically relating to a method for the preservation and preparation of highly antioxidant active sea buckthorn protoplasm exosomes and their application. Background Technology

[0002] Sea buckthorn is a unique medicinal and edible economic plant in my country. Its fruit is rich in a variety of natural antioxidant active components such as flavonoids, polyphenols, tocopherols, and unsaturated fatty acids. It has the biological activity of scavenging free radicals, inhibiting oxidative stress, and repairing cellular oxidative damage. It has important application and development value in the fields of food, cosmetics, and biomedicine.

[0003] Sea buckthorn exosomes are 30-150 nm nanoscale extracellular vesicles naturally secreted from sea buckthorn fruit, capable of specifically encapsulating the core antioxidant components of sea buckthorn. Compared to free antioxidant active components, sea buckthorn exosomes have significant advantages such as good biocompatibility, strong cell penetration, stable sustained release of active ingredients, and high bioavailability. They are high-performance natural antioxidant functional carriers with high development and application value.

[0004] Current mainstream preparation technologies for sea buckthorn exosomes generally suffer from process defects. Existing preparation schemes mostly rely on process routes such as room temperature high-speed ultracentrifugation, high-temperature vacuum concentration, and organic solvent extraction. In actual production and product applications, these methods have revealed multiple technical drawbacks: the strong mechanical shear force generated during high-speed ultracentrifugation can tear the exosome vesicle structure, causing the antioxidant active ingredients encapsulated inside the vesicles to leak out and directly reduce the content of effective ingredients in the product; high-temperature concentration treatment can cause irreversible degradation of heat-sensitive antioxidant components such as flavonoids and polyphenols, significantly weakening the antioxidant bioactivity of the product; and organic solvent extraction processes pose a risk of chemical reagent residues, failing to meet the safety and compliance requirements for food, cosmetics, and biopharmaceutical excipients, severely limiting the expansion of application scenarios for sea buckthorn exosome products.

[0005] Meanwhile, existing technologies have not designed a dedicated stabilization system to protect the active components of sea buckthorn exosomes, which is a key factor in their vesicle structure and physicochemical properties. This makes the exosomes prone to vesicle collapse and oxidative deactivation of active components during the entire preparation process and subsequent storage. The resulting sea buckthorn exosome products generally suffer from low purity, poor bioactivity, weak batch-to-batch quality stability, and short shelf life at room temperature, failing to meet the actual demands of high-quality mass production in industrial applications.

[0006] A comprehensive search of domestic and international patent literature, core academic journals, and publicly available technical materials revealed that existing technologies do not disclose an integrated, mild preparation process that couples low-temperature gradient impurity removal, low-pressure staged ultrafiltration, dedicated isotonic protection, and low-temperature freeze-drying. Furthermore, no technical solutions have been found that utilize a mild, end-to-end preparation process to directionally retain the high antioxidant activity of sea buckthorn exosomes. In summary, existing technologies cannot simultaneously solve the multiple technical challenges of vesicle structure damage, antioxidant activity attenuation, poor product storage stability, insufficient safety and compliance, and difficulty in large-scale mass production during the preparation of sea buckthorn exosomes. Summary of the Invention

[0007] The purpose of this invention is to provide a method for the preservation and preparation of highly antioxidant sea buckthorn protoplasm exosomes and their applications. It can be widely used in many fields such as skin antioxidant repair agents, antioxidant functional health foods, and excipients for drugs that repair oxidative stress damage in the body. The product has a wide range of applicable scenarios and outstanding application value and market potential.

[0008] The objective of this invention is achieved through the following technical solution: This invention provides a method for preserving and preparing sea buckthorn protoplasm exosomes with high antioxidant activity, comprising the following steps: (1) Low-temperature sterile pretreatment to remove impurities: Select fresh, mature sea buckthorn fruits that are free from rot, mold, and pests and diseases. Wash and drain them with sterile pure water at 0-4℃. Juice them at low speed under sterile low-temperature conditions at 4-8℃ to obtain crude sea buckthorn pulp. Place the crude sea buckthorn pulp in a 4℃ low-temperature centrifuge and use a gradient low-speed centrifugation process to remove large solid particles and oil phase impurities in sequence. Collect the middle layer of clear sterile aqueous filtrate. (2) Low temperature and low pressure fractional ultrafiltration enrichment: The clarified filtrate obtained in step (1) was filtered through 0.45μm and 0.22μm sterile microporous membranes to remove impurities. Then, it was concentrated by ultrafiltration using a 100-150kDa ultrafiltration membrane, constant temperature of 2-6℃ and low pressure of 0.1-0.3MPa. The membrane retentate was collected to obtain the crude extract of sea buckthorn exosomes. (3) Isotonic steady-state protection treatment: Mix the crude exosome extract obtained in step (2) with sterile isotonic activity protection buffer at a volume ratio of 1:3 to 1:5, and incubate at 4°C in the dark for 30 to 60 minutes to stabilize the exosome vesicle membrane structure and inhibit the degradation and loss of antioxidant active components. (4) Low-temperature vacuum freeze-drying: The revitalized mixture is pre-frozen at -40 to -50℃ for 2 to 4 hours, and then freeze-dried under vacuum at 10 to 30 Pa for 12 to 18 hours. It is then sealed and stored to obtain freeze-dried powder of sea buckthorn pulp exosomes with high antioxidant activity.

[0009] Furthermore, the gradient low-speed centrifugation process described in step (1) is as follows: first, centrifuge at 3000-4000 r / min for 10-15 min to remove pulp fiber residue, and then centrifuge at 6000-8000 r / min for 15-20 min to achieve separation of the three phases of oil phase, water phase and solid impurities. The centrifugation temperature is kept constant at 4℃ throughout the process.

[0010] Furthermore, in step (2), an ultrafiltration membrane with a molecular weight cutoff of 120 kDa is used, the ultrafiltration temperature is kept constant at 4°C, and the ultrafiltration pressure is kept constant at 0.2 MPa.

[0011] Furthermore, the sterile isotonic active protective buffer solution mentioned in step (3) is a phosphate buffer system with a pH of 7.0 to 7.4, containing 0.85% to 0.95% sodium chloride, 0.02% to 0.05% sodium ascorbate, and 0.01% to 0.03% trehalose by mass.

[0012] Furthermore, after the freeze-drying in step (4) is completed, the product is filled and sealed with sterile high-purity nitrogen and stored in a light-proof and airtight environment at -20℃. The antioxidant activity decay rate of the product is ≤8% after 6 months of storage at room temperature.

[0013] The present invention also provides a highly antioxidant active sea buckthorn protoplast exosome, which is prepared by the aforementioned retention preparation method, and the particle size distribution of the sea buckthorn exosome is 30-150 nm.

[0014] Furthermore, the sea buckthorn exosomes exhibit a vesicle integrity retention rate of ≥92%, a DPPH free radical scavenging rate of ≥88%, an ABTS free radical scavenging rate of ≥90%, and a superoxide anion scavenging rate of ≥85%.

[0015] This invention also provides the application of the aforementioned highly antioxidant active sea buckthorn pulp exosomes in the preparation of products with antioxidant and repair effects.

[0016] Furthermore, the product with antioxidant and repairing effects is a skin antioxidant and repairing cosmetic, and the sea buckthorn exosome freeze-dried powder is added to the cosmetic matrix at a mass ratio of 0.5% to 1%.

[0017] Furthermore, the product with antioxidant and repairing effects is an antioxidant functional health food or a pharmaceutical excipient for repairing oxidative stress damage in the body.

[0018] The beneficial effects of this invention are as follows: This invention employs a fully low-temperature, purely physical coupling preparation process, avoiding the strong mechanical shear damage caused by high-speed ultracentrifugation, the degradation of heat-sensitive components due to high-temperature concentration, and the chemical reagent interference caused by organic solvent extraction. Throughout the entire preparation process, the complete vesicle structure of sea buckthorn exosomes is protected, locking in the core antioxidant active components such as flavonoids, polyphenols, and vitamins encapsulated within. Compared to traditional preparation processes, this method achieves a vesicle integrity retention rate of ≥92%, effectively preventing leakage and loss of active ingredients caused by vesicle rupture. The product's ability to scavenge DPPH free radicals, ABTS free radicals, and superoxide anion free radicals is significantly improved, with overall antioxidant activity increased by more than 40% compared to traditional processes, fully leveraging the bioactive advantages of sea buckthorn exosomes as natural antioxidant nanocarriers.

[0019] This invention employs a coupled purification pathway combining gradient low-speed centrifugation for impurity removal and staged low-pressure ultrafiltration for enrichment. This process removes fruit pulp fiber residue, oily phase impurities, and fine particulate matter step by step, effectively improving the purity and particle size uniformity of exosome products. Simultaneously, it incorporates a proprietary isotonic activity protection buffer system adapted to the physiological environment of sea buckthorn exosomes, along with low-temperature vacuum freeze-drying and aseptic sealing storage processes. This stabilizes the exosome vesicle membrane protein structure and inhibits the oxidative degradation of heat-sensitive antioxidant components. This addresses the technical pain points of traditional processes, such as structural collapse, oxidative inactivation, and short storage periods. The antioxidant activity attenuation rate of the finished product after 6 months of room temperature storage is less than 8%, demonstrating significantly superior storage stability compared to existing technologies. It exhibits minimal batch-to-batch performance fluctuations and is suitable for long-distance transportation and storage in various scenarios.

[0020] The entire preparation process of this invention does not involve the addition of any organic solvents, eliminating the risk of chemical reagent residues. The resulting sea buckthorn exosomes are mild, non-irritating, and biocompatible, fully meeting the safety access standards for cosmetics, functional foods, and biopharmaceutical excipients. Compared to free sea buckthorn antioxidant components, exosome vesicle carriers possess stronger cell penetration and sustained-release effects, resulting in higher bioavailability. They can be widely applied in various fields such as skin antioxidant repair preparations, antioxidant functional health foods, and excipients for drugs repairing oxidative stress damage. The products are suitable for a wide range of scenarios, demonstrating significant application value and market potential.

[0021] The process of this invention is clear, the parameters are controllable, and the repeatability is strong. All processes can be completed using general-purpose biological preparation equipment, without relying on specialized and expensive equipment such as high-speed ultracentrifuges, which greatly reduces production input costs and operational barriers. Compared with the limitations of traditional high-speed ultracentrifugation processes, such as low output, small processing capacity, and poor batch stability, this process can achieve continuous and stable industrial-scale production. Moreover, the limited range of process parameters has good fault tolerance. Fluctuations in normal parameters during production will not significantly affect product performance. The batch stability of mass production is strong, and it has extremely strong industrial applicability and industrialization promotion value. Detailed Implementation

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0024] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0025] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0026] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0027] The method for preserving and preparing high antioxidant activity sea buckthorn exosomes described in this invention follows a fully low-temperature, purely physical coupled process route, consisting of low-temperature aseptic pretreatment for impurity removal, low-temperature and low-pressure fractional ultrafiltration enrichment, isotonic steady-state preservation treatment, and low-temperature vacuum freeze-drying. The entire preparation process avoids high-temperature degradation, strong mechanical shearing, and chemical reagent intervention, thereby achieving high preservation of the vesicle structure and core antioxidant active components of sea buckthorn exosomes.

[0028] Example 1 This embodiment provides a method for preserving and preparing sea buckthorn protoplasm exosomes with high antioxidant activity. The specific operation steps are as follows: (1) Low-temperature sterile pretreatment for impurity removal: Fresh, mature sea buckthorn fruit without rot, mold, or pests was selected as raw material. The fresh fruit was washed three times with sterile pure water at 0-4℃ to remove surface dust and impurities. The surface water was drained. The juice was extracted by low-speed cell wall breaking under sterile low-temperature environment at 6℃ to obtain uniform crude sea buckthorn pulp. The crude sea buckthorn pulp was transferred to a 4℃ constant temperature centrifuge and impurity removal was carried out by gradient low-speed centrifugation: First, centrifugation was carried out at 3500r / min for 12min to remove large particles of pulp and fiber residue. Then, centrifugation was carried out at 7000r / min for 18min to achieve three-phase separation of the upper oil phase, the middle clear water phase, and the bottom solid precipitate. The middle clear sterile water phase filtrate was collected.

[0029] (2) Low-temperature and low-pressure fractional ultrafiltration enrichment: The clarified filtrate obtained in step (1) was filtered sequentially through 0.45μm and 0.22μm sterile microporous membranes to further remove fine impurities and microbial contamination. Then, an ultrafiltration membrane with a molecular weight cutoff of 120kDa was used to concentrate the filtrate under constant temperature of 4℃ and constant low pressure of 0.2MPa. The retentate of the ultrafiltration membrane was collected to obtain the purified crude extract of sea buckthorn exosomes.

[0030] (3) Isotonic steady-state protection treatment: The crude extract of sea buckthorn exosomes obtained in step (2) is mixed with sterile isotonic active protection buffer at a volume ratio of 1:4. The sterile isotonic active protection buffer is a phosphate buffer system with a pH of 7.2, containing 0.9% sodium chloride, 0.03% sodium ascorbate and 0.02% trehalose by mass. The mixture is placed in a low temperature and light-proof environment at 4°C for 45 min to stabilize the exosome vesicle membrane structure and inhibit the degradation and loss of heat-sensitive antioxidant active components such as flavonoids and polyphenols.

[0031] (4) Low-temperature vacuum freeze-drying and shaping: The mixture after the protection treatment is placed in a -45℃ environment for 3 hours for pre-freezing, and then transferred to a vacuum freeze-drying equipment. It is vacuum freeze-dried for 15 hours under a vacuum of 20Pa to obtain sea buckthorn pulp exosome freeze-dried powder; the finished product is filled and sealed with sterile high-purity nitrogen and stored in a light-proof and airtight environment at -20℃.

[0032] Example 2 This embodiment provides a method for preserving and preparing sea buckthorn protoplasm exosomes with high antioxidant activity. The specific operation steps are as follows: (1) Low-temperature sterile pretreatment to remove impurities: Fresh, mature sea buckthorn fruit without rot, mold, or pests was selected as raw material. The fruit was washed three times with sterile pure water at 0-4℃. After draining the surface water, the juice was extracted at low speed under sterile low-temperature environment at 4℃ to obtain crude sea buckthorn pulp. The crude sea buckthorn pulp was placed in a 4℃ constant temperature centrifuge. First, it was centrifuged at 3000r / min for 15min to remove large particles of pulp and fiber residue. Then, it was centrifuged at 6000r / min for 20min to achieve three-phase separation. The middle layer of clear sterile aqueous filtrate was collected.

[0033] (2) Low temperature and low pressure fractional ultrafiltration enrichment: The clarified filtrate obtained in step (1) was filtered through 0.45μm and 0.22μm sterile microporous membranes to remove impurities. Then, an ultrafiltration membrane with a molecular weight cutoff of 100kDa was used to concentrate the filtrate under constant temperature of 2℃ and constant low pressure of 0.1MPa. The membrane retentate was collected to obtain the crude extract of sea buckthorn exosomes.

[0034] (3) Isotonic steady-state protection treatment: The crude extract of sea buckthorn exosomes and sterile isotonic active protection buffer were mixed evenly at a volume ratio of 1:3; the sterile isotonic active protection buffer was a phosphate buffer system with a pH of 7.0, containing 0.85% sodium chloride, 0.02% sodium ascorbate and 0.01% trehalose by mass concentration; the mixture was incubated in a low temperature and light-proof environment at 4°C for 30 min.

[0035] (4) Low-temperature vacuum freeze-drying and shaping: The thawed mixture was pre-frozen in a -40℃ environment for 4 hours, and then vacuum freeze-dried for 18 hours under a vacuum of 10Pa to obtain sea buckthorn pulp exosome freeze-dried powder; the finished product was filled and sealed with sterile high-purity nitrogen and stored in a light-proof and airtight environment at -20℃.

[0036] Example 3 This embodiment provides a method for preserving and preparing sea buckthorn protoplasm exosomes with high antioxidant activity. The specific operation steps are as follows: (1) Low-temperature sterile pretreatment to remove impurities: Fresh, mature sea buckthorn fruit without rot, mold, or pests was selected as raw material. The fruit was washed three times with sterile pure water at 0-4℃. After draining the surface water, the juice was extracted at low speed under sterile low-temperature environment at 8℃ to obtain crude sea buckthorn pulp. The crude sea buckthorn pulp was placed in a 4℃ constant temperature centrifuge. First, it was centrifuged at 4000r / min for 10min to remove large particles of pulp and fiber residue. Then, it was centrifuged at 8000r / min for 15min to achieve three-phase separation. The middle layer of clear sterile aqueous filtrate was collected.

[0037] (2) Low temperature and low pressure fractional ultrafiltration enrichment: The clarified filtrate obtained in step (1) was filtered through 0.45μm and 0.22μm sterile microporous membranes to remove impurities. Then, an ultrafiltration membrane with a molecular weight cutoff of 150kDa was used to concentrate the filtrate under constant temperature of 6℃ and constant low pressure of 0.3MPa. The membrane retentate was collected to obtain the crude extract of sea buckthorn exosomes.

[0038] (3) Isotonic steady-state protection treatment: The crude extract of sea buckthorn exosomes and sterile isotonic active protection buffer were mixed evenly at a volume ratio of 1:5; the sterile isotonic active protection buffer was a phosphate buffer system with a pH of 7.4, containing 0.95% sodium chloride, 0.05% sodium ascorbate and 0.03% trehalose by mass concentration; the mixture was incubated in a low temperature and light-protected environment at 4°C for 60 min.

[0039] (4) Low temperature vacuum freeze drying and shaping: The reviving mixture is pre-frozen in an environment of -50℃ for 2 hours, and then vacuum freeze-dried in a vacuum of 30Pa for 12 hours to obtain sea buckthorn pulp exosome freeze-dried powder; the finished product is filled and sealed with sterile high-purity nitrogen and stored in a light-proof and airtight environment of -20℃.

[0040] Comparative Example 1 Using fresh sea buckthorn fruit of the same quality and quantity as in Example 1 as raw material, sea buckthorn exosome powder was prepared using traditional methods: the cell walls were broken and juiced at room temperature, and impurities were removed by high-speed centrifugation at 12000 r / min for 30 min at room temperature. After concentration by ultrafiltration at room temperature, the sample was naturally air-dried to obtain a sea buckthorn exosome control sample.

[0041] Experiment Example 1: Product Performance Comparison Test Using standardized national testing methods, the samples prepared in Examples 1, 2, and 3 were compared with the control sample in Comparative Example 1 in terms of vesicle structure integrity, antioxidant activity, and storage stability. The specific test results are shown in Table 1 below: Table 1 Comparison of Product Performance

[0042] As shown in the table above, the product performance differences between the process of this invention and the traditional process are as follows: the vesicle integrity retention rate and the three types of free radical scavenging rate of each embodiment of this invention are significantly higher than those of the traditional process, and the activity decay rate after 6 months of storage at room temperature is much lower than that of the traditional process.

[0043] The parallel experimental data above show that, within the parameter range defined in the claims of this invention, the sea buckthorn exosomes prepared in each embodiment all exhibit extremely high vesicle integrity retention rate, excellent antioxidant activity, and storage stability, with performance indicators far superior to those of traditional preparation processes. Among these, the product exhibits the best overall performance under the parameter conditions of Example 1, representing the optimal implementation parameters for this invention. Examples 2 and 3 verify the universality and stability of the process parameter range of this invention; fine-tuning the parameters within the range does not significantly reduce product performance, demonstrating high process tolerance and strong mass production stability, fully proving the scientific validity and practicality of the technical solution of this invention.

[0044] Application Example 1: Application of Skin Antioxidant Repair Cosmetics The sea buckthorn exosome freeze-dried powder prepared in this invention can be added to skin care bases such as lotions, serums, and creams at a mass ratio of 0.5% to 1%. It can effectively remove skin free radicals, repair ultraviolet and oxidative damage, and improve dull and aging skin. The product is gentle and non-irritating, suitable for all skin types, and especially suitable for the antioxidant repair of sensitive skin.

[0045] Application Example 2: Application of Antioxidant Functional Health Foods The sea buckthorn exosome freeze-dried powder prepared by this invention can be used as a natural functional additive in beverages, solid beverages, meal replacement foods and other products to prepare natural antioxidant functional foods, which can help alleviate oxidative stress damage in the human body and enhance the body's antioxidant capacity.

[0046] Application Example 3: Application of excipients in drugs for repairing oxidative stress damage The sea buckthorn exosomes prepared by this invention have excellent biocompatibility and can be used as core excipients for oxidative stress damage repair preparations and drugs to improve the efficiency of cellular oxidative damage repair and enhance drug efficacy.

[0047] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for preserving and preparing sea buckthorn protoplasm exosomes with high antioxidant activity, characterized in that, Includes the following steps: (1) Low-temperature sterile pretreatment to remove impurities: Select fresh, mature sea buckthorn fruits that are free from rot, mold, and pests and diseases. Wash and drain them with sterile pure water at 0-4℃. Juice them at low speed under sterile low-temperature conditions at 4-8℃ to obtain crude sea buckthorn pulp. Place the crude sea buckthorn pulp in a 4℃ low-temperature centrifuge and use a gradient low-speed centrifugation process to remove large solid particles and oil phase impurities in sequence. Collect the middle layer of clear sterile aqueous filtrate. (2) Low temperature and low pressure fractional ultrafiltration enrichment: The clarified filtrate obtained in step (1) was filtered through 0.45μm and 0.22μm sterile microporous membranes to remove impurities. Then, it was concentrated by ultrafiltration using a 100-150kDa ultrafiltration membrane, constant temperature of 2-6℃ and low pressure of 0.1-0.3MPa. The membrane retentate was collected to obtain the crude extract of sea buckthorn exosomes. (3) Isotonic steady-state protection treatment: Mix the crude exosome extract obtained in step (2) with sterile isotonic activity protection buffer at a volume ratio of 1:3 to 1:5, and incubate at 4°C in the dark for 30 to 60 minutes to stabilize the exosome vesicle membrane structure and inhibit the degradation and loss of antioxidant active components. (4) Low-temperature vacuum freeze-drying: The revitalized mixture is pre-frozen at -40 to -50℃ for 2 to 4 hours, and then freeze-dried under vacuum at 10 to 30 Pa for 12 to 18 hours. It is then sealed and stored to obtain freeze-dried powder of sea buckthorn pulp exosomes with high antioxidant activity.

2. The method for preserving and preparing highly antioxidant sea buckthorn protoplasm exosomes according to claim 1, characterized in that, The gradient low-speed centrifugation process described in step (1) is as follows: first, centrifuge at 3000-4000 r / min for 10-15 min to remove pulp fiber residue, and then centrifuge at 6000-8000 r / min for 15-20 min to achieve separation of the three phases of oil phase, water phase and solid impurities. The centrifugation temperature is kept constant at 4℃ throughout the process.

3. The method for preparing and preserving highly antioxidant sea buckthorn protoplast exosomes according to claim 1, characterized in that, In step (2), an ultrafiltration membrane with a molecular weight cutoff of 120 kDa is used, the ultrafiltration temperature is kept constant at 4°C, and the ultrafiltration pressure is kept constant at 0.2 MPa.

4. The method for preparing and preserving highly antioxidant sea buckthorn protoplast exosomes according to claim 1, characterized in that, The sterile isotonic active protective buffer solution mentioned in step (3) is a phosphate buffer system with pH 7.0 to 7.4, containing sodium chloride with a mass concentration of 0.85% to 0.95%, sodium ascorbate with a mass concentration of 0.02% to 0.05%, and trehalose with a mass concentration of 0.01% to 0.03%.

5. The method for preparing and preserving highly antioxidant sea buckthorn protoplast exosomes according to claim 1, characterized in that, After freeze-drying in step (4), the product is filled and sealed with sterile high-purity nitrogen and stored in a light-proof and airtight environment at -20℃. The antioxidant activity decay rate of the product is ≤8% after 6 months of storage at room temperature.

6. A highly antioxidant-active sea buckthorn protoplasm exosome, characterized in that, The sea buckthorn exosomes are prepared by the retention preparation method according to any one of claims 1 to 5, and the particle size distribution of the sea buckthorn exosomes is 30 to 150 nm.

7. The highly antioxidant active sea buckthorn protoplast exosomes according to claim 6, characterized in that, The sea buckthorn exosomes exhibited a vesicle integrity retention rate of ≥92%, a DPPH free radical scavenging rate of ≥88%, an ABTS free radical scavenging rate of ≥90%, and a superoxide anion scavenging rate of ≥85%.

8. The application of a highly antioxidant active sea buckthorn pulp exosome as described in claim 6 or 7 in the preparation of products with antioxidant and repair effects.

9. The application according to claim 8, characterized in that, The product with antioxidant and repairing effects is a skin antioxidant and repairing cosmetic, and the sea buckthorn exosome freeze-dried powder is added to the cosmetic matrix at a mass ratio of 0.5% to 1%.

10. The application according to claim 8, characterized in that, The products with antioxidant and repair effects are antioxidant functional health foods or excipients for drugs that repair oxidative stress damage in the body.

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