An efficient extraction method of exosomes from ginseng callus, exosomes and applications thereof

Abstract

The present invention relates to the field of plant tissue extraction technology, and in particular to a method for efficiently extracting exosomes from ginseng callus and its application. The method comprises: S1, mixing ginseng callus with PBS buffer, homogenizing, and filtering to obtain a filtrate; S2, adding a protease inhibitor and a deep eutectic solvent to the filtrate, mixing well, and then allowing to stand to obtain a mixed solution; S3, centrifuging and filtering the mixed solution to retain the supernatant; S4, ultracentrifuging the supernatant, collecting the precipitate, and resuspending it with PBS to obtain a resuspension; S5, further filtering the resuspension to obtain a filtrate, and storing it at low temperature. The ginseng callus exosomes extracted by the extraction method of the present invention can be used to prepare medicines or cosmetics with antioxidant, anti-inflammatory, and anti-aging effects, effectively solving the technical problems of low extraction efficiency, insufficient purity, complex extraction operation, and poor stability of the extracted exosomes in existing exosome extraction technologies.

Description

Technical Field

[0001] The present invention relates to the technical field of plant tissue extraction, and in particular to a method for efficiently extracting exosomes from ginseng callus, the exosomes, and applications thereof. Background Art

[0002] Ginseng is a traditional Chinese medicinal herb. Pharmacopoeias record its sweet and bitter taste, with a slightly warming effect. It enters the spleen, lung, heart, and kidney meridians, boasting benefits such as tonifying qi and blood, calming the mind, and improving intelligence. Modern research indicates that the active ingredients in ginseng exosomes, such as ginsenosides, microRNA, and antioxidant enzymes, exhibit significant potential in anti-inflammatory, antioxidant, and tissue repair activities. These natural ingredients are readily absorbed by the human body and pose no risk of immune rejection, offering promising applications in new drug development, skincare and beauty, health and wellness, and functional foods.

[0003] In recent years, ginseng callus has become an important source for obtaining highly active exosomes (vesicles with a diameter of approximately 30-150 nm) due to its scalable culture and stable metabolites. The cell wall structure of ginseng callus is complex, and its surface lipid composition differs significantly from that of animal-derived exosomes. Plant cell walls contain large amounts of cellulose and pectin, making it difficult to efficiently release exosomes and remove impurities using conventional methods. The functional activity of ginseng exosomes is highly dependent on their contents (such as ginsenosides). Existing extraction methods (such as ultracentrifugation and polymer precipitation) rely on multiple centrifugation steps, which are not only time-consuming and have low extraction yields, but also easily disrupt the integrity of the exosome membrane structure due to high shear forces, resulting in reduced exosome functionality and compromising subsequent storage, delivery, and application. Currently, there is no mature technology for extracting exosomes from ginseng callus tissue. Existing exosome extraction technologies have problems such as low efficiency, insufficient purity, and complex operation. The stability of the extracted exosomes is also poor. Long-term high-speed centrifugation can easily lead to rupture of the exosome membrane structure and loss of functional activity. After ultracentrifugation, exosome aggregation affects its interaction with target cells. The above problems have seriously restricted the application of ginseng callus tissue exosomes in medical and cosmetic fields.

[0004] Patent publication number CN115400061A discloses a method for extracting ginseng callus tissue, which increases the ginsenoside content in the final extract through organic solvent pretreatment followed by enzymatic hydrolysis. Patent publication number CN118853531A discloses exosome-like vesicles from ginseng tubers and their preparation method, extracting exosomes via filtration and ultracentrifugation. These patents only examine the exosome contents and extraction process, but do not address the content, purity, or stability of the extracted exosomes. Summary of the Invention

[0005] To address the shortcomings of the existing technology, the present invention aims to provide a highly efficient method for extracting exosomes from ginseng callus. This method, coupled with a "filtration + organic solvent + extraction centrifugation" technique, achieves efficient and stable extraction of exosomes from ginseng callus. The ginseng callus exosomes extracted using this method can be used to prepare pharmaceuticals or cosmetics with antioxidant, anti-inflammatory, and anti-aging effects. This method effectively addresses the technical issues of existing exosome extraction technologies, such as low extraction efficiency, insufficient purity, complex extraction procedures, and the poor stability and aggregation of extracted exosomes.

[0006] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0007] A technical solution of the present invention provides a method for efficiently extracting exosomes from ginseng callus, comprising the following steps:

[0008] S1, mixing ginseng callus with PBS buffer, homogenizing, and filtering to obtain a filtrate;

[0009] S2, adding a protease inhibitor and a deep eutectic solvent to the filtrate of step S1, mixing and then allowing to stand to obtain a mixed solution;

[0010] S3, centrifuging and filtering the mixed solution after standing in step S2, and retaining the supernatant;

[0011] S4, ultracentrifuging the supernatant obtained in step S3, collecting the precipitate after filtration, and resuspending it in PBS to obtain a resuspension;

[0012] S5, further filtering the resuspension obtained in step S4, and the filtrate is a solution containing ginseng callus-derived exosomes, which is stored at low temperature.

[0013] In some possible embodiments, in step S1, the mass ratio of ginseng callus tissue to PBS buffer is 1:(8-12).

[0014] Furthermore, in step S1, the pore size of the filter membrane used for filtration is 3-10 μm.

[0015] In some possible embodiments, in step S2, the protease inhibitor is any one of 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, bestatin, leupeptin, and pepstatin A.

[0016] Protease inhibitors can be used to inhibit the activity of endogenous proteases in ginseng callus tissue, preventing the degradation of exosome membrane proteins and other contents. On the one hand, this maintains the structural integrity and functional activity of the exosomes. On the other hand, it also prevents the degradation products of exosome membrane proteins and other contents from mixing into the preparation, thereby achieving a purification effect.

[0017] In some possible embodiments, the protease inhibitor is 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride and leupeptin at a mass ratio of (2-4):1.

[0018] In some possible embodiments, in step S2, the deep eutectic solvent consists of a hydrogen bond donor and a hydrogen bond acceptor.

[0019] In some possible embodiments, the molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is 1:(2-5).

[0020] In some possible embodiments, the hydrogen bond donor is any one of organic acids and alcohols.

[0021] For example, the hydrogen bond donor may be vitamin C, lactic acid or maltose.

[0022] In some possible embodiments, the hydrogen bond acceptor is any one of choline chloride, betaine, and urea.

[0023] Preferably, the deep eutectic solvent is vitamin C-deep eutectic solvent.

[0024] Deep eutectic solvents (DES) are eutectic mixtures formed by intermolecular interactions between hydrogen bond donors and hydrogen bond acceptors upon heating. Vitamin C (ascorbic acid), a reducing organic acid, can act as a hydrogen bond donor. Vitamin C-deep eutectic solvents are prepared by combining vitamin C with choline chloride. Vitamin C-deep eutectic solvents share the natural properties of ascorbic acid and possess a high number of hydroxyl groups in their molecular structure, which enhances their ability to form hydrogen bonds with exosomes. Deep eutectic solvents can dissolve polysaccharides (such as cellulose and pectin) in plant cell walls, disrupting cell wall integrity. Combined with the inhibitory effect of protease inhibitors, they can reduce the co-solubility of polysaccharides and proteins, minimizing impurity contamination and improving exosome purity.

[0025] In addition, the sugar chains of ginsenosides (such as Rb1 and Rg1) are rich in hydroxyl groups, which form hydrogen bonds with components in deep eutectic solvents (such as hydroxyl groups of ascorbic acid and polar groups of choline chloride), resulting in an adsorption effect on ginsenosides, which can enhance the solubility of ginsenosides in the solvent and thus improve the extraction rate of exosomes.

[0026] In terms of stability, ascorbic acid in the deep eutectic solvent contains three hydroxyl groups (C2, C3, and C6) and one carboxylic acid group (C1). Under neutral pH conditions, the hydroxyl group (-OH) forms hydrogen bonds with polar groups (such as amino or carboxylic acid groups) on the phospholipid layer on the surface of exosomes, adsorbs on the surface of exosomes, and partially dissociates into carboxylate groups (-COOH) bound to the carboxylic acid groups. -), carrying negative charges to supplement the negative charge density, increasing the Zeta potential and enhancing the repulsive force between particles, thereby improving potential stability and avoiding exosome aggregation.

[0027] In some possible embodiments, in step S2, the deep eutectic solvent is obtained by the following preparation method:

[0028] (1) Weighing a hydrogen bond donor and a hydrogen bond acceptor, and mixing them evenly to obtain a mixed raw material;

[0029] (2) heating and stirring the mixed raw materials of step (1) to form a transparent and uniform liquid;

[0030] (3) The liquid obtained in step (2) is cooled to room temperature to obtain a deep eutectic solvent.

[0031] In some possible embodiments, in step (1), the molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is 1:(2-5).

[0032] In some possible embodiments, in step (2), the heating temperature is 70-90°C.

[0033] In some possible embodiments, in step S2, the mass fraction of the protease inhibitor in the filtrate of step S1 is 0.5-2%.

[0034] Preferably, in step S2, the mass fraction of the protease inhibitor in the filtrate of step S1 is 0.8-2%.

[0035] In some possible implementations, in step S2, the mass fraction of the deep eutectic solvent in the filtrate of step S1 is 0.3-1%.

[0036] Preferably, in step S2, the mass fraction of the deep eutectic solvent in the filtrate of step S1 is 0.4-1%.

[0037] Furthermore, in step S2, the standing time is 10-20 minutes.

[0038] In some possible embodiments, step S3 specifically includes: centrifuging and filtering the mixed solution after standing in step S2 at a centrifugal force of 300-500g, 600-1000g, and 10000-20000g, respectively, with each centrifugation time being 10-30min and the centrifugation temperature being 4°C. After each centrifugation, taking the supernatant and filtering it.

[0039] Illustratively, in step S3, the centrifugal force of the first centrifugation can be selected as 300g, 400g or 500g, and the centrifugation time can be selected as 10min, 15min or 20min; the centrifugal force of the second centrifugation can be selected as 600g, 800g or 1000g, and the centrifugation time can be selected as 10min, 15min or 20min; the centrifugal force of the third centrifugation can be selected as 10000g, 15000g or 20000g, and the centrifugation time can be selected as 10min, 15min, 20min, 25min or 30min.

[0040] Preferably, in step S3, the mixed solution after standing in step S2 is centrifuged at a centrifugal force of 350-450 g, 550-950 g, and 10,000-18,000 g for 15-25 min respectively.

[0041] Preferably, in step S3, the mixed solution after standing in step S2 is centrifuged at a centrifugal force of 300 g, 600 g, and 10,000 g for 20 min respectively.

[0042] In step S3, the first and second centrifugation and filtration are used to remove ginseng callus cell fragments, intact cells and large particle impurities (precipitates), and the exosomes and smaller vesicles are retained in the supernatant after centrifugation and filtration.

[0043] In step S3, the third centrifugation and filtration are used to remove medium-sized impurities (precipitates) such as apoptotic bodies and platelets, and to further purify the supernatant. The supernatant after centrifugation and filtration contains exosomes and smaller vesicles.

[0044] Furthermore, in step S3, the pore size of the filter membrane used for filtration is 0.6-1.0 μm;

[0045] In some possible embodiments, in step S4, the centrifugal force used for ultracentrifugation is 80,000-120,000 g, the centrifugation time is 0.5-1 h, and the centrifugation temperature is 4°C.

[0046] Preferably, in step S4, the centrifugal force used for ultracentrifugation is 90,000-110,000 g, the centrifugation time is 0.7-1 h, and the centrifugation temperature is 4°C.

[0047] Illustratively, in step S4, the centrifugal force used for ultracentrifugation is 80,000 g, 100,000 g, or 120,000 g, and the centrifugation time is 0.5 h or 1 h.

[0048] Preferably, in step S4, the centrifugal force used for ultracentrifugation is 100,000 g, and the centrifugation time is 1 h.

[0049] In step S4, the role of ultracentrifugation is to precipitate exosomes. After filtration, the precipitate contains purified exosomes, and the supernatant contains residual proteins and low-density particles.

[0050] In some possible implementations, in step S4, the pore size of the filtration membrane is 0.4-0.8 μm.

[0051] Furthermore, in step S5, the pore size of the filter membrane is 0.2-0.22 μm.

[0052] The resuspension in step S4 mainly contains the purified exosomes, which are present in a dispersed state in the solution. The purpose of further filtration in step S5 is to remove cell debris and proteins that were not completely removed during the centrifugation process.

[0053] Furthermore, in step S5, the final product containing ginseng callus-derived exosomes is placed in a 4°C temperature environment and stored at low temperature.

[0054] The present invention also provides exosomes extracted by the efficient extraction method of ginseng callus-derived exosomes according to any of the above schemes, and their use in the preparation of cosmetics, medicines, and health foods with antioxidant, anti-aging, and / or anti-inflammatory functions.

[0055] The present invention has the following beneficial effects:

[0056] (1) The present invention is a method for extracting exosomes from ginseng callus tissue. By adding protease inhibitors and deep eutectic solvents, the destruction of the exosome structure is avoided, the co-dissolution of polysaccharides and proteins is reduced, and impurity contamination is reduced. Combined with the centrifugation process, the extracted exosomes have high purity, higher ginsenoside content, and excellent stability.

[0057] (2) The present invention can dissolve polysaccharides (such as cellulose, pectin, etc.) in plant cell walls through deep eutectic solvents, destroying the integrity of the cell walls, thereby efficiently releasing exosomes; the effective components in the deep eutectic solvents can form hydrogen bonds with the hydroxyl-rich sugar chains of ginsenosides (such as Rb1, Rg1), resulting in an adsorption effect on ginsenosides, enhancing the solubility of ginsenosides in the solvent, and increasing the extraction content and stability.

[0058] (3) The deep eutectic solvent in the present invention can also form hydrogen bonds with the polar groups of the phospholipid layer on the surface of exosomes, adsorbing on the surface of exosomes and increasing the negative charge density through the carboxylic acid group, thereby increasing the Zeta potential and enhancing the repulsive force between particles, thereby improving the potential stability and avoiding exosome aggregation.

[0059] (4) The extraction method of the present invention can improve the integrity, purity and stability of the prepared exosomes. The process is simple, the cost is low, and the exosome extraction efficiency is high. The extracted ginseng callus exosomes have good application prospects in the preparation of cosmetics, drugs or health foods with antioxidant, anti-aging and anti-inflammatory functions. DETAILED DESCRIPTION

[0060] The following describes the embodiments of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Although the present invention will be described in conjunction with the preferred embodiments, this does not mean that the features of the present invention are limited to the embodiments.

[0061] The cell wall structure of ginseng callus is complex, and its surface lipid composition differs significantly from that of animal-derived exosomes. Plant cell walls contain a large amount of cellulose and pectin, making it difficult to efficiently release intracellular exosomes and remove impurities using conventional methods. The functional activity of ginseng exosomes is highly dependent on their contents (such as ginsenosides). Existing extraction methods (such as ultracentrifugation and polymer precipitation) rely on multiple centrifugation steps, which are not only time-consuming and have low extraction rates, but are also easily destroyed by high shear forces to maintain the integrity of the exosome membrane structure, resulting in reduced exosome functionality and affecting subsequent storage, delivery, and application.

[0062] Based on the above problems, the present invention proposes an efficient extraction method for exosomes from ginseng callus tissue. Ginseng callus tissue is used as raw material, PBS buffer is added and homogenized, and then pretreated with protease inhibitors and deep eutectic solvents (DESs) before centrifugation and filtration. This achieves efficient and stable extraction of exosomes from ginseng callus tissue.

[0063] Example

[0064] The embodiments of the present invention provide a method for efficiently extracting exosomes from ginseng callus, comprising the following steps:

[0065] S1, mixing ginseng callus with PBS buffer, homogenizing, and filtering to obtain a filtrate;

[0066] S2, adding a protease inhibitor and a deep eutectic solvent to the filtrate of step S1, mixing and then allowing to stand to obtain a mixed solution;

[0067] S3, centrifuging and filtering the mixed solution after standing in step S2, and retaining the supernatant;

[0068] S4, ultracentrifuging the supernatant obtained in step S3, collecting the precipitate, and resuspending it in PBS to obtain a resuspension;

[0069] S5, further filtering the resuspended filtrate obtained in step S4 to obtain the final product, which is stored at low temperature.

[0070] In some embodiments, in step S1, the mass ratio of ginseng callus tissue to PBS buffer is 1:(8-12).

[0071] In some embodiments, in step S1, the pore size of the filter membrane used for filtration is 3-10 μm.

[0072] In some embodiments, in step S2, the protease inhibitor is any one of 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, bestatin, leupeptin, and pepstatin A.

[0073] In some embodiments, in step S2, the deep eutectic solvent consists of a hydrogen bond donor and a hydrogen bond acceptor.

[0074] In some embodiments, in step S2, the molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is 1:(2-5).

[0075] In some embodiments, the hydrogen bond donor is any one of organic acids and alcohols.

[0076] For example, the hydrogen bond donor may be vitamin C, lactic acid or maltose.

[0077] In some embodiments, the hydrogen bond acceptor is any one of choline chloride, betaine, and urea.

[0078] Preferably, the deep eutectic solvent is vitamin C-deep eutectic solvent.

[0079] In some embodiments, in step S2, the deep eutectic solvent is obtained by the following preparation method:

[0080] (1) Weighing a hydrogen bond donor and a hydrogen bond acceptor, and mixing them evenly to obtain a mixed raw material;

[0081] (2) heating and stirring the mixed raw materials of step (1) to form a transparent and uniform liquid;

[0082] (3) The liquid obtained in step (2) is cooled to room temperature to obtain a deep eutectic solvent.

[0083] In some possible embodiments, in step (1), the molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is 1:(2-5).

[0084] In some embodiments, in step (2), the heating temperature is 70-90°C.

[0085] In some embodiments, in step S2, the mass fraction of the protease inhibitor in the filtrate of step S1 is 0.5-2%;

[0086] In some embodiments, in step S2, the mass fraction of the protease inhibitor in the filtrate of step S1 is 0.8-2%.

[0087] In some embodiments, in step S2, the mass fraction of the deep eutectic solvent in the filtrate of step S1 is 0.3-1%.

[0088] In some embodiments, in step S2, the mass fraction of the deep eutectic solvent in the filtrate of step S1 is 0.4-1%.

[0089] In some embodiments, in step S2, the standing time is 10-20 minutes.

[0090] In some embodiments, step S3 specifically includes: centrifuging the mixed solution after standing in step S2 three times at a centrifugal force of 300-500g, 600-1000g, and 10000-20000g, each centrifugation time is 10-20min, and the centrifugation temperature is 4°C. After each centrifugation, the mixture is filtered with a filter membrane with a pore size of 0.6-1.0μm, and the supernatant is taken.

[0091] Illustratively, in step S3, the centrifugal force of the first centrifugation can be selected as 300g, 400g or 500g, and the centrifugation time can be selected as 10min, 15min or 20min; the centrifugal force of the second centrifugation can be selected as 600g, 800g or 1000g, and the centrifugation time can be selected as 10min, 15min or 20min; the centrifugal force of the third centrifugation can be selected as 10000g, 15000g or 20000g, and the centrifugation time can be selected as 10min, 15min, 20min, 25min or 30min.

[0092] In some embodiments, in step S3, the mixed solution after standing in step S2 is centrifuged at a centrifugal force of 350-450 g, 550-950 g, and 10,000-18,000 g for 15-25 min, respectively.

[0093] In some embodiments, in step S3, the mixed solution after standing in step S2 is centrifuged at a centrifugal force of 300 g, 600 g, and 10,000 g for 20 minutes respectively.

[0094] In some embodiments, in step S4, the centrifugal force used for ultracentrifugation is 80,000-120,000 g, and the centrifugation time is 0.5-1 h.

[0095] Illustratively, in step S4, the centrifugal force used for ultracentrifugation is 80,000 g, 100,000 g, or 120,000 g, and the centrifugation time is 0.5 h or 1 h.

[0096] In some embodiments, in step S4, the centrifugal force used for ultracentrifugation is 90,000-110,000 g, the centrifugation time is 0.7-1 h, and the centrifugation temperature is 4°C.

[0097] In some embodiments, in step S4, the centrifugal force used for ultracentrifugation is 100,000 g, and the centrifugation time is 1 hour.

[0098] In some embodiments, in step S4, the pore size of the filter membrane used for filtration is 0.4-0.8 μm.

[0099] In some embodiments, in step S5, the pore size of the filter membrane used for filtration is 0.2-0.22 μm.

[0100] In some embodiments, in step S5, the final product containing ginseng callus-derived exosomes is placed in a 4° C. temperature environment and stored at low temperature.

[0101] Other embodiments of the present invention also provide the use of exosomes extracted by the efficient extraction method of ginseng callus-derived exosomes according to any of the above schemes in the preparation of cosmetics, medicines, and health foods with antioxidant, anti-aging, and / or anti-inflammatory functions.

[0102] To make the purpose, technical solutions and advantages of the present invention clearer, the embodiments of the present invention are described in further detail below. Unless otherwise specified, the raw materials used in the following examples are all common commercially available products, and the reagents are analytically pure reagents.

[0103] Example 1

[0104] This embodiment provides a method for efficiently extracting exosomes from ginseng callus, comprising the following steps:

[0105] S1, washing ginseng callus tissue, mixing it with PBS buffer at a mass ratio of 1:10, homogenizing it using a homogenizer, and filtering it through a filter membrane with a pore size of 5 μm to obtain a filtrate;

[0106] S2, adding 1% by mass of a protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride and 0.5% by mass of a deep eutectic solvent to the filtrate, mixing well, and then standing for 10 minutes to obtain a mixed solution;

[0107] The deep eutectic solvent is obtained by the following preparation method:

[0108] (1) mixing vitamin C and choline chloride in a molar ratio of 1:3 to obtain a mixed raw material;

[0109] (2) Heat the mixed raw materials at 80°C and stir until a transparent and uniform liquid is gradually formed;

[0110] (3) Cooling the obtained transparent liquid to room temperature to obtain a deep eutectic solvent;

[0111] S3, centrifuging the mixture after standing at 4°C at 400g, 800g, and 15,000g for 20 min respectively. After each centrifugation, filter the mixture with a 0.8 μm pore size filter membrane and retain the supernatant.

[0112] S4, the supernatant after the third centrifugation was ultracentrifuged at a centrifugal force of 100,000 g for 1 h, filtered through a filter membrane with a pore size of 0.45 μm, and the precipitate was collected. The precipitate was resuspended in PBS buffer at a mass ratio of 1:10 to obtain a resuspension;

[0113] S5. Filter the resuspension using a filter membrane with a pore size of 0.2 μm. The filtrate is a solution containing exosomes derived from ginseng callus tissue, which is stored at 4°C.

[0114] Example 2

[0115] This embodiment provides a method for efficiently extracting exosomes from ginseng callus, comprising the following steps:

[0116] S1, washing ginseng callus tissue, mixing it with PBS buffer at a mass ratio of 1:10, homogenizing it using a homogenizer, and filtering it through a filter membrane with a pore size of 5 μm to obtain a filtrate;

[0117] S2, adding 2% by mass of a protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride and 1% by mass of a deep eutectic solvent to the filtrate, mixing well, and then standing for 10 minutes to obtain a mixed solution;

[0118] The deep eutectic solvent is obtained by the following preparation method:

[0119] (1) mixing vitamin C and choline chloride in a molar ratio of 1:3 to obtain a mixed raw material;

[0120] (2) Heat the mixed raw materials at 80°C and stir until a transparent and uniform liquid is gradually formed;

[0121] (3) Cooling the obtained transparent liquid to room temperature to obtain a deep eutectic solvent;

[0122] S3, centrifuging the mixture after standing at 4°C at 400g, 800g, and 15,000g for 20 min respectively, filtering with a 0.8 μm pore size filter membrane after each centrifugation, and retaining the supernatant;

[0123] S4, ultracentrifuging the supernatant after the third centrifugation at a centrifugal force of 100,000 g for 1 h, filtering through a filter membrane with a pore size of 0.45 μm, collecting the precipitate, and resuspending the precipitate in PBS buffer at a mass ratio of 1:10 to obtain a resuspension;

[0124] S5. Filter the resuspension using a filter membrane with a pore size of 0.2 μm. The filtrate is a solution containing exosomes derived from ginseng callus tissue, which is stored at 4°C.

[0125] Example 3

[0126] This embodiment provides a method for efficiently extracting exosomes from ginseng callus, comprising the following steps:

[0127] S1, washing ginseng callus tissue, mixing it with PBS buffer at a mass ratio of 1:10, homogenizing it using a homogenizer, and filtering it through a filter membrane with a pore size of 5 μm to obtain a filtrate;

[0128] S2, adding 0.8% by mass of protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride and 0.4% by mass of deep eutectic solvent to the filtrate, mixing and standing for 10 minutes to obtain a mixed solution;

[0129] The deep eutectic solvent is obtained by the following preparation method:

[0130] (1) mixing vitamin C and choline chloride in a molar ratio of 1:3 to obtain a mixed raw material;

[0131] (2) Heat the mixed raw materials at 80°C and stir until a transparent and uniform liquid is gradually formed;

[0132] (3) Cooling the obtained transparent liquid to room temperature to obtain a deep eutectic solvent;

[0133] S3, centrifuging the mixture after standing at 4°C at 400g, 800g, and 15,000g for 20 min respectively. After each centrifugation, filter the mixture with a 0.8 μm pore size filter membrane and retain the supernatant.

[0134] S4, the supernatant after the third centrifugation was ultracentrifuged at a centrifugal force of 100,000 g for 1 h, filtered through a filter membrane with a pore size of 0.45 μm, and the precipitate was collected. The precipitate was resuspended in PBS buffer at a mass ratio of 1:10 to obtain a resuspension;

[0135] S5. Filter the resuspension using a filter membrane with a pore size of 0.2 μm. The filtrate is a solution containing exosomes derived from ginseng callus tissue, which is stored at 4°C.

[0136] Example 4

[0137] This embodiment provides a method for efficiently extracting exosomes from ginseng callus, comprising the following steps:

[0138] S1, washing ginseng callus tissue, mixing it with PBS buffer at a mass ratio of 1:10, homogenizing it using a homogenizer, and filtering it through a filter membrane with a pore size of 5 μm to obtain a filtrate;

[0139] S2, adding 0.5% by mass of protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride and 0.3% by mass of deep eutectic solvent to the filtrate, mixing and standing for 10 minutes to obtain a mixed solution;

[0140] The deep eutectic solvent is obtained by the following preparation method:

[0141] (1) mixing vitamin C and choline chloride in a molar ratio of 1:3 to obtain a mixed raw material;

[0142] (2) Heat the mixed raw materials at 80°C and stir until a transparent and uniform liquid is gradually formed;

[0143] (3) Cooling the obtained transparent liquid to room temperature to obtain a deep eutectic solvent;

[0144] S3, centrifuging the mixture after standing at 4°C at 400g, 800g, and 15,000g for 20 min respectively, filtering with a 0.8 μm pore size filter membrane after each centrifugation, and retaining the supernatant;

[0145] S4, the supernatant after the third centrifugation was ultracentrifuged at a centrifugal force of 100,000 g for 1 h, filtered through a filter membrane with a pore size of 0.45 μm, and the precipitate was collected. The precipitate was resuspended in PBS buffer at a mass ratio of 1:10 to obtain a resuspension;

[0146] S5. Filter the resuspension using a filter membrane with a pore size of 0.2 μm. The filtrate is a solution containing exosomes derived from ginseng callus tissue, which is stored at 4°C.

[0147] Example 5

[0148] This embodiment provides a method for efficiently extracting exosomes from ginseng callus, comprising the following steps:

[0149] S1, washing ginseng callus tissue, mixing it with PBS buffer at a mass ratio of 1:10, homogenizing it using a homogenizer, and filtering it through a filter membrane with a pore size of 5 μm to obtain a filtrate;

[0150] S2, adding 1% by mass of a protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride and 0.5% by mass of a deep eutectic solvent to the filtrate, mixing well, and then standing for 10 minutes to obtain a mixed solution;

[0151] The deep eutectic solvent is obtained by the following preparation method:

[0152] (1) mixing vitamin C and choline chloride in a molar ratio of 1:3 to obtain a mixed raw material;

[0153] (2) Heat the mixed raw materials at 80°C and stir until a transparent and uniform liquid is gradually formed;

[0154] (3) Cooling the obtained transparent liquid to room temperature to obtain a deep eutectic solvent;

[0155] S3, centrifuging the mixture after standing at 4°C at 500g, 1000g, and 20,000g for 20 min respectively. After each centrifugation, filter the mixture through a 0.8 μm pore size filter membrane and retain the supernatant.

[0156] S4, the supernatant after the third centrifugation was ultracentrifuged at a centrifugal force of 120,000 g for 1 h, filtered through a filter membrane with a pore size of 0.45 μm, and the precipitate was collected. The precipitate was resuspended in PBS buffer at a mass ratio of 1:10 to obtain a resuspension;

[0157] S5. Filter the resuspension using a filter membrane with a pore size of 0.2 μm. The filtrate is a solution containing exosomes derived from ginseng callus tissue, which is stored at 4°C.

[0158] Example 6

[0159] This embodiment provides a method for efficiently extracting exosomes from ginseng callus, comprising the following steps:

[0160] S1, washing ginseng callus tissue, mixing it with PBS buffer at a mass ratio of 1:10, homogenizing it using a homogenizer, and filtering it through a filter membrane with a pore size of 5 μm to obtain a filtrate;

[0161] S2, adding 1% by mass of a protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride and 0.5% by mass of a deep eutectic solvent to the filtrate, mixing well, and then standing for 10 minutes to obtain a mixed solution;

[0162] The deep eutectic solvent is obtained by the following preparation method:

[0163] (1) mixing vitamin C and choline chloride in a molar ratio of 1:3 to obtain a mixed raw material;

[0164] (2) Heat the mixed raw materials at 80°C and stir until a transparent and uniform liquid is gradually formed;

[0165] (3) Cooling the obtained transparent liquid to room temperature to obtain a deep eutectic solvent;

[0166] S3, centrifuging the mixture after standing at 4°C at 500g, 1000g, and 20,000g for 10 min respectively, filtering with a 0.8 μm pore size filter membrane after each centrifugation, and retaining the supernatant;

[0167] S4, the supernatant after the third centrifugation was ultracentrifuged at a centrifugal force of 120,000 g for 0.5 h, filtered through a filter membrane with a pore size of 0.45 μm, and the precipitate was collected. The precipitate was resuspended with PBS buffer at a mass ratio of 1:10 to obtain a resuspension;

[0168] S5. Filter the resuspension using a filter membrane with a pore size of 0.2 μm. The filtrate is a solution containing exosomes derived from ginseng callus tissue, which is stored at 4°C.

[0169] Example 7

[0170] This embodiment provides a method for efficiently extracting exosomes from ginseng callus, comprising the following steps:

[0171] S1, washing ginseng callus tissue, mixing it with PBS buffer at a mass ratio of 1:10, homogenizing it using a homogenizer, and filtering it through a filter membrane with a pore size of 5 μm to obtain a filtrate;

[0172] S2, adding 1% by mass of a protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride and 0.5% by mass of a deep eutectic solvent to the filtrate, mixing well, and then standing for 10 minutes to obtain a mixed solution;

[0173] The deep eutectic solvent is obtained by the following preparation method:

[0174] (1) mixing vitamin C and choline chloride in a molar ratio of 1:3 to obtain a mixed raw material;

[0175] (2) Heat the mixed raw materials at 80°C and stir until a transparent and uniform liquid is gradually formed;

[0176] (3) Cooling the obtained transparent liquid to room temperature to obtain a deep eutectic solvent;

[0177] S3, centrifuging the mixture after standing at 4°C at 400g, 800g, and 15,000g for 10 min respectively, filtering with a 0.8 μm pore size filter membrane after each centrifugation, and retaining the supernatant;

[0178] S4, ultracentrifuging the supernatant after the third centrifugation at a centrifugal force of 100,000 g for 0.5 h, filtering through a filter membrane with a pore size of 0.45 μm, collecting the precipitate, and resuspending the precipitate in PBS buffer at a mass ratio of 1:10 to obtain a resuspension;

[0179] S5. Filter the resuspension using a filter membrane with a pore size of 0.2 μm. The filtrate is a solution containing exosomes derived from ginseng callus tissue, which is stored at 4°C.

[0180] Example 8

[0181] This embodiment provides a method for efficiently extracting exosomes from ginseng callus, comprising the following steps:

[0182] S1, washing ginseng callus tissue, mixing it with PBS buffer at a mass ratio of 1:10, homogenizing it using a homogenizer, and filtering it through a filter membrane with a pore size of 5 μm to obtain a filtrate;

[0183] S2, adding 1% by mass of a protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride and 0.5% by mass of a deep eutectic solvent to the filtrate, and then standing for 10 minutes to obtain a mixed solution;

[0184] The deep eutectic solvent is obtained by the following preparation method:

[0185] (1) mixing vitamin C and choline chloride in a molar ratio of 1:3 to obtain a mixed raw material;

[0186] (2) Heat the mixed raw materials at 80°C and stir until a transparent and uniform liquid is gradually formed;

[0187] (3) Cooling the obtained transparent liquid to room temperature to obtain a deep eutectic solvent;

[0188] S3, centrifuging the mixture after standing at 4°C at 300g, 600g, and 10,000g for 20 min respectively. After each centrifugation, filter the mixture through a 0.8 μm pore size filter membrane and retain the supernatant.

[0189] S4, ultracentrifuging the supernatant after the third centrifugation at a centrifugal force of 100,000 g for 1 h, filtering through a filter membrane with a pore size of 0.45 μm, collecting the precipitate, and resuspending the precipitate in PBS buffer at a mass ratio of 1:10 to obtain a resuspension;

[0190] S5. Filter the resuspension using a filter membrane with a pore size of 0.2 μm. The filtrate is a solution containing exosomes derived from ginseng callus tissue, which is stored at 4°C.

[0191] Example 9

[0192] This example is basically the same as Example 1, except that leupeptin is used as the protease inhibitor instead of 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, and the amount of the protease inhibitor added remains unchanged.

[0193] Example 10

[0194] This example is basically the same as Example 1, except that the protease inhibitor is a mixture of 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride and leupeptin in a mass ratio of 3:1, instead of a single type of 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, and the amount of protease inhibitor added remains unchanged.

[0195] Comparative Example 1

[0196] This comparative example provides a method for efficiently extracting exosomes from ginseng callus, comprising the following steps:

[0197] S1, washing ginseng callus tissue, mixing it with PBS buffer at a mass ratio of 1:10, homogenizing it using a homogenizer, and filtering it through a filter membrane with a pore size of 5 μm to obtain a filtrate;

[0198] S2, centrifuge the filtrate at 4°C at 400g, 800g, and 15,000g for 20 min, respectively. After each centrifugation, filter through a 0.8 μm pore membrane and retain the supernatant.

[0199] S3, the supernatant after the third centrifugation was ultracentrifuged at a centrifugal force of 100,000 g for 1 h, filtered through a filter membrane with a pore size of 0.45 μm, and the precipitate was collected. The precipitate was mixed with PBS buffer at a mass ratio of 1:10 to obtain a resuspension;

[0200] S4. Filter the resuspension through a filter membrane with a pore size of 0.2 μm. The filtrate is a solution containing exosomes derived from ginseng callus tissue, which is stored at 4°C.

[0201] Comparative Example 2

[0202] This embodiment provides a method for efficiently extracting exosomes from ginseng callus, comprising the following steps:

[0203] S1, washing ginseng callus tissue, mixing it with PBS buffer at a mass ratio of 1:10, homogenizing it using a homogenizer, and filtering it through a filter membrane with a pore size of 5 μm to obtain a filtrate;

[0204] S2, adding 1% by mass of protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride to the filtrate and letting it stand for 10 minutes to obtain a mixed solution;

[0205] S3, centrifuging the mixture after standing at 4°C at 400g, 800g, and 15,000g for 20 min respectively, filtering with a 0.8 μm pore size filter membrane after each centrifugation and retaining the supernatant;

[0206] S4, the supernatant after the third centrifugation was ultracentrifuged at a centrifugal force of 100,000 g for 1 h, filtered through a filter membrane with a pore size of 0.45 μm, and the precipitate was collected. The precipitate was resuspended in PBS buffer at a mass ratio of 1:10 to obtain a resuspension;

[0207] S5. Filter the resuspension using a filter membrane with a pore size of 0.2 μm. The filtrate is a solution containing exosomes derived from ginseng callus tissue, which is stored at 4°C.

[0208] Comparative Example 3

[0209] This embodiment provides a method for efficiently extracting exosomes from ginseng callus, comprising the following steps:

[0210] S1, washing ginseng callus tissue, mixing it with PBS buffer at a mass ratio of 1:10, homogenizing it using a homogenizer, and filtering it through a filter membrane with a pore size of 5 μm to obtain a filtrate;

[0211] S2, adding 0.5% by mass of a deep eutectic solvent to the filtrate and letting it stand for 10 minutes to obtain a mixed solution;

[0212] The deep eutectic solvent is obtained by the following preparation method:

[0213] (1) mixing vitamin C and choline chloride in a molar ratio of 1:3 to obtain a mixed raw material;

[0214] (2) Heat the mixed raw materials at 80°C and stir until a transparent and uniform liquid is gradually formed;

[0215] (3) Cooling the obtained transparent liquid to room temperature to obtain a deep eutectic solvent;

[0216] S3, centrifuging the mixture after standing at 4°C at 400g, 800g, and 15,000g for 20 min respectively. After each centrifugation, filter the mixture through a 0.8 μm pore size filter membrane and retain the supernatant.

[0217] S4, the supernatant after the third centrifugation was ultracentrifuged at a centrifugal force of 100,000 g for 1 h, filtered through a filter membrane with a pore size of 0.45 μm, and the precipitate was collected. The precipitate was resuspended in PBS buffer at a mass ratio of 1:10 to obtain a resuspension;

[0218] S5. Filter the resuspension using a filter membrane with a pore size of 0.2 μm. The filtrate is a solution containing exosomes derived from ginseng callus tissue, which is stored at 4°C.

[0219] Performance Testing

[0220] The performance tests of the solutions (final products) containing ginseng callus-derived exosomes extracted in Examples 1-10 and Comparative Examples 1-3 were performed as follows: the ginsenoside content in the exosome solution was detected by high-performance liquid chromatography; the potential stability of the exosome solution was tested using a Zeta potential detector; and the purity of the exosome content in the exosome solution was detected using a nanoparticle size analyzer. The test results are shown in Table 1.

[0221] Among them, the method for calculating the purity of exosomes is: first detect the total number of vesicles A contained in the exosome solution, and then calculate the number of vesicles B with a size between 30-150 nm. The purity of exosomes is the ratio of B to A.

[0222] Table 1 Properties of ginseng callus-derived exosome solution

[0223]

[0224] By analyzing the data in Table 1 and comparing Example 1 with Comparative Examples 1-3, it can be seen that the addition of either a protease inhibitor or a deep eutectic solvent alone can appropriately increase the amount of ginsenoside precipitation, potential stability, and exosome purity. However, the simultaneous addition of a protease inhibitor and a deep eutectic solvent in Example 1 for synergistic effect can not only avoid the destruction of the exosome structure and reduce the co-solubility of polysaccharides and proteins, but also improve the degree of solubility of the ginseng callus cell wall and increase the adsorption and solubility of ginsenosides, thereby significantly improving the amount of ginsenoside precipitation, potential stability, and exosome purity.

[0225] Comparing the results of Examples 1-3, it can be seen that in Example 2, when the ratio of protease inhibitor and deep eutectic solvent was increased to 2% and 1%, respectively, the optimization efficiency of ginsenoside precipitation, potential stability, and exosome purity decreased. This may be because the amount of protease inhibitor and deep eutectic solvent in the system exceeded the optimal dosage value, that is, the effect of adding more amount decreased. In Example 3, when lower content of protease inhibitor and deep eutectic solvent was added, the amount of ginsenoside precipitation decreased, but it could still be maintained at a high level. Compared with Examples 1-3, when the ratio of protease inhibitor and deep eutectic solvent was reduced to 0.5% and 0.3%, respectively, in Example 4, the amount of ginsenoside precipitation and exosome purity decreased significantly, probably due to insufficient amount of protease inhibitor and deep eutectic solvent. Therefore, when the ratio of protease inhibitor and deep eutectic solvent was 0.8-2% and 0.4-1%, respectively, the exosome solution had a higher ginsenoside content, better potential stability, and higher exosome purity.

[0226] Comparing the results of Example 1 and Examples 5-8, it can be seen that changing the centrifugal force and centrifugation time has little effect on the ginsenoside content; in Example 5, increasing the centrifugal force may cause the exosomes to aggregate during the centrifugation process, thereby reducing the potential stability, and may damage the exosome structure to produce impurities (such as damaged membrane proteins, etc.), thereby reducing the purity. Therefore, during the exosome extraction process, the centrifugal force should not be too large; in Example 6, the centrifugal force is the same as that of Example 5, and the centrifugation time is too short, which affects the precipitation rate of ginsenosides and the purity of exosomes, but is better than Example 5; and compared with Example 1, Example 7 has the same centrifugal force and shortened centrifugation time, resulting in a decrease in all data, indicating that the appropriate centrifugal force is suitable for a relatively long centrifugation time; in Example 8, the centrifugal force decreased for all three times, resulting in insufficient centrifugal strength, so the yield of ginsenosides and the purity of exosomes decreased.

[0227] Comparing the results of Example 1 with Examples 9-10, it can be seen that the addition of different types of protease inhibitors in step S2 has different effects on the ginsenoside content, potential stability, and purity of the extracted exosomes. Comparing Example 1 with Example 9, the results show that using 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride as a protease inhibitor is superior to using leupeptin in terms of ginsenoside content, potential stability, and exosome purity. Comparing Example 1 with Example 10, the results show that the combination of two protein inhibitors is more effective than using a single type. In Example 10, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride and leupeptin in a mass ratio of 3:1 were combined, and the ginsenoside content, potential stability, and purity performance were slightly higher than those of Example 1.

[0228] Application Examples

[0229] The anti-aging test was conducted using the ginseng callus-derived exosome solution extracted using the extraction methods of Examples 1-10 and Comparative Examples 1-3. The specific contents are as follows:

[0230] (1) 150 female mice were selected, with an average age of 3 months and an average weight of 25 g;

[0231] (2) 150 mice were randomly divided into 15 groups, with 10 mice in each group;

[0232] (3) Grouping: 1 control group, 1 aging group; the remaining 13 experimental groups were gavaged with the exosome solutions extracted from Examples 1-10 and Comparative Examples 1-3;

[0233] (4) Test method:

[0234] The mice in the control group were injected subcutaneously with an equal volume of sterile saline at the back of the neck every day;

[0235] Mice in the aging group were injected subcutaneously with 12.5% ​​D-galactose (1000 mg / kg) at the back of the neck daily and gavaged with sterile saline (100 mg / kg) daily;

[0236] The mice in the experimental group were injected subcutaneously with 12.5% ​​D-galactose (1000 mg / kg) at the back of the neck daily, and were gavaged with exosome solution (100 mg / kg) daily.

[0237] The above experimental process lasted for 30 days. After 30 days, all mice were treated with the sudden cervical death method, and the SOD value in the mouse blood was measured using a superoxide dismutase kit (superoxide dismutase SOD has antioxidant and anti-aging effects). The results were averaged. The test results are shown in Table 2 below.

[0238] Table 2 SOD values ​​in the blood of mice in each group

[0239]

[0240] By analyzing the data in Table 2, compared with the SOD values ​​of the aging group, the decline in the SOD values ​​of the mice in each example group after galactose aging modeling was alleviated to varying degrees, indicating that the exosome solution extracted by the callus tissue exosome extraction method of the present invention has a certain anti-aging effect.

[0241] The present invention combines protease inhibitors with deep eutectic solvents, adjusts and optimizes the centrifugal force and centrifugation time, and achieves efficient extraction of exosomes by short-term, low-speed centrifugation, avoiding functional damage to the exosomes caused by membrane structure damage due to high-speed centrifugation. The obtained exosome solution has a good anti-aging effect.

[0242] Although the preferred embodiments of the present invention have been disclosed above, they are not intended to limit the present invention. Any person skilled in the art can make possible changes and modifications to the technical solutions of the present invention by using the methods and technical contents disclosed above without departing from the spirit and scope of the present invention. Therefore, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solutions of the present invention shall fall within the scope of protection of the technical solutions of the present invention.

Claims

1. A method for efficiently extracting exosomes from ginseng callus, characterized in that: The following steps are involved: S1, mixing ginseng callus with PBS buffer, homogenizing, and filtering to obtain a filtrate; S2, adding a protease inhibitor and a deep eutectic solvent to the filtrate of step S1, mixing and then allowing to stand to obtain a mixed solution; S3, centrifuging and filtering the mixed solution after standing in step S2, and retaining the supernatant; S4, ultracentrifuging the supernatant obtained in step S3, collecting the precipitate after filtration, and resuspending it in PBS to obtain a resuspension; S5, further filtering the resuspension obtained in step S4, and the filtrate is a solution containing ginseng callus-derived exosomes; The protease inhibitor is any one of 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride and leupeptin; The deep eutectic solvent is a vitamin C-choline chloride deep eutectic solvent; The mass fraction of the protease inhibitor in the filtrate of step S1 is 0.5-2%, and the mass fraction of the deep eutectic solvent in the filtrate of step S1 is 0.3-1%.

2. The efficient extraction method of exosomes from ginseng callus according to claim 1, characterized in that: In step S1, the mass ratio of the ginseng callus tissue to the PBS buffer solution is 1:(8-12), and the filtration uses a filter membrane with a pore size of 3-10 μm.

3. The efficient extraction method of exosomes from ginseng callus according to claim 1, characterized in that: In step S2, the standing time is 10-20 minutes; The deep eutectic solvent consists of a hydrogen bond donor and a hydrogen bond acceptor; The hydrogen bond donor is vitamin C; The hydrogen bond acceptor is choline chloride; The molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is 1:(2-5).

4. The efficient extraction method of exosomes from ginseng callus according to claim 1, characterized in that: In step S2, the deep eutectic solvent is obtained by the following preparation method: (1) Weighing a hydrogen bond donor and a hydrogen bond acceptor, and mixing them evenly to obtain a mixed raw material; Wherein, the hydrogen bond donor is vitamin C, and the hydrogen bond acceptor is choline chloride; (2) heating and stirring the mixed raw materials of step (1) to form a transparent and uniform liquid; (3) The liquid obtained in step (2) is cooled to room temperature to obtain a deep eutectic solvent.

5. The efficient extraction method of exosomes from ginseng callus according to claim 4, characterized in that: The heating temperature in step (2) is 70-90°C.

6. The efficient extraction method of exosomes from ginseng callus according to claim 1, characterized in that: In step S2, the mass fraction of the protease inhibitor in the filtrate of step S1 is 0.8-2%, and the mass fraction of the deep eutectic solvent in the filtrate of step S1 is 0.4-1%.

7. The method for efficiently extracting exosomes from ginseng callus according to claim 1, characterized in that: The step S3 specifically includes: centrifuging the mixed solution after standing in step S2 at a centrifugal force of 300-500g, 600-1000g, and 10000-20000g in sequence, taking the supernatant and filtering it after each centrifugation, the centrifugation time for each time is 10-30min, the centrifugation temperature is 4°C, and the filtration uses a filter membrane with a pore size of 0.6-1.0μm.

8. The method for efficiently extracting exosomes from ginseng callus according to claim 1, characterized in that: In step S4, the centrifugal force used in the ultracentrifugation is 80,000-120,000 g, the centrifugation time is 0.5-1 h, the centrifugation temperature is 4° C., and the filtration uses a filter membrane with a pore size of 0.4-0.8 μm; In step S5, the filtration uses a filter membrane with a pore size of 0.2-0.22 μm.

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