CN116726090A - Application of extracellular vesicles of Chinese wolfberry in promoting tissue repair or growth - Google Patents

Abstract

The invention relates to an application of medlar extracellular vesicles (GqDNV) in preparing medicaments for promoting tissue repair or growth; the tissue is skeletal muscle; the contents of the extracellular vesicles of Lycium barbarum comprise saccharides. The invention provides a method for separating GqDNV from fresh Chinese wolfberry stably and efficiently, and the extracted extracellular vesicles of the Chinese wolfberry are easy to be biologically absorbed and utilized, so that the problem of poor bioavailability of oral Chinese wolfberry is solved. GqDNV is used as an intervening substance to have the effects of promoting tissue repair/growth and the like, and the GqDNV is used as a drug carrier, so that the GqDNV has the effects of improving the exercise function of a mice with wilted muscles and promoting skeletal muscle growth, and can be applied to the medical field. Compared with wolfberry fruits or extracted wolfberry polysaccharide and the like, the GqDNV has similar biological activity and better bioavailability, can be directly absorbed by tissue cells in a intramuscular injection mode and the like, and has good medical application prospect.

Description

Application of extracellular vesicles of Chinese wolfberry in promoting tissue repair or growth

Technical Field

The invention relates to the field of biological agents, in particular to application of medlar extracellular vesicles in promoting tissue repair or growth.

Background

Medlar is a traditional Chinese herbal medicine, and in the compendium of materia medica, it is recorded that medlar has the functions of strengthening tendons and bones, resisting fatigue and aging. At present, researches have reported that the main active ingredient, namely, lycium barbarum polysaccharide, extracted from the medlar has the biological activity effects of nourishing liver and kidney, improving eyesight, delaying aging, enhancing immunity, reducing blood sugar and the like. Lycium barbarum polysaccharide is one of the main active components of Lycium barbarum, and generally consists of six monosaccharides (galactose, glucose, rhamnose, arabinose, mannose and xylose) and antioxidant. The preparation process of the wolfberry extract comprises the following steps: dried wolfberry fruit (200 g) was boiled in 2 liters of water for 3 hours, filtered with filter paper, and placed in a vacuum rotary evaporator at 60 ℃. And then dried in a freeze dryer at a temperature of-80℃and a pressure of 500mTorr. The obtained fructus Lycii extract was dissolved in PBS and stored at 4deg.C. The preparation method of the wolfberry extract is similar to that of wolfberry polysaccharide. At present, the application of medlar is mostly direct oral administration or water decoction. In addition, the wolfberry extract or wolfberry polysaccharide is prepared through distillation and freeze drying, and the effective action of the wolfberry extract or wolfberry polysaccharide is researched through mouse and cell experiments. For example: the medlar extract and the betaine which is the biological active component thereof can promote the differentiation and energy metabolism of C2C12 cells by increasing the phosphorylation level of AMP activated protein kinase and acetyl coenzyme a carboxylase, which suggests that the medlar extract is helpful for preventing skeletal muscle dysfunction; meanwhile, the lycium barbarum polysaccharide is proved to play an anti-fatigue role by improving lipid peroxidation level of skeletal muscle tissues of sub-healthy mice and increasing antioxidant enzyme activity of the skeletal muscle tissues; the fructus Lycii water decoction extract can also increase the quality of mouse gastrocnemius and tibialis anterior, and increase average running distance of mouse by increasing the proportion of mouse IIa type oxidized muscle fiber.

Extracellular vesicles refer to vesicles with membrane structures that are peeled off from the cell membrane or secreted by the cell, exist outside the cell, and have the functions of transferring encapsulated bioactive molecules (including DNA, RNA, proteins, and lipids), promoting intercellular communication, and regulating pathological processes. Extracellular vesicles are considered to be a promising therapeutically active substance and drug delivery vehicle due to their proven pharmacological activity, satisfactory biocompatibility, specific tissue targeting and good drug delivery capacity. The isolation of plant-derived extracellular vesicles from animal-derived extracellular vesicles is simple, high in yield, wide in source, and remarkable in activity, and has been attracting attention in recent years. The form and composition of the plant extracellular vesicles are similar to those of animal extracellular vesicles, and the plant extracellular vesicles have functions similar to those of the source plants, and play an important role in non-cell autonomous mode and even information exchange among species.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide the application of the extracellular vesicles of the medlar, which have high bioavailability, can improve the absorption and utilization degree of tissues in a intramuscular injection mode and the like, can promote the growth of skeletal muscle and muscle when being applied to the repair of skeletal muscle, and have good medical application prospect.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

in a first aspect, the present invention provides the use of an extracellular vesicle of lycium barbarum in the manufacture of a medicament for promoting tissue repair or growth; the contents of the extracellular vesicles of Lycium barbarum comprise saccharides.

Preferably, the tissue is skeletal muscle; the tissue repair or growth is manifested by an increase in grip strength or an increase in skeletal muscle fiber cross-sectional area.

Wolfberry is a traditional Chinese herbal medicine, and in 1987, wolfberry is classified as a homologous species of medicine and food by the Ministry of health of China. It is recorded in Ben Cao gang mu (compendium of materia Medica) in Lishizhen that matrimony vine can strengthen tendons and bones and has anti-fatigue and anti-aging effects. The wolfberry water decoction and wolfberry polysaccharide also prove to play a role in promoting the recovery of skeletal muscle injury and skeletal muscle growth of mice. However, the prior medlar and the extracted products thereof are mostly used for oral administration, have lower bioavailability and smaller application range. Therefore, it is desired to extract extracellular vesicles (GqDNV) from wolfberry fruits, and to search for a wider range of uses of wolfberry fruits while making the bioactive components in wolfberry fruits more easily absorbed and utilized by tissue cells.

The GqDNV extracted by the invention has the effects of improving the muscle function of the skeletal muscle of the mice and promoting the growth of the skeletal muscle, but further action mechanisms are still under study. Compared with extracts of Lycium barbarum fruits, lycium barbarum polysaccharides and the like, the GqDNV has high bioavailability, can improve the absorption and utilization degree of tissues by intramuscular injection and the like, and has good medical application prospect. Meanwhile, the GqDNV serving as the extracellular vesicle of plant source has the advantages of wide source and high extraction rate, is easy to be absorbed by cells, and has potential as a drug carrier.

Preferably, the saccharide comprises at least one of sucrose, glucose, D-fructose, D-cellobiose, maltose, D-mannose, 1, 6-anhydro- β -D-glucose, trehalose, methyl β -D-galactopyranoside, D-arabinose, D-galacturonic acid, L-rhamnose, D-xylulose and arabitol.

As shown by GqDNV polysaccharide targeting metabolome results, sugar contained in GqDNV comprises sucrose, glucose, D-fructose, D-cellobiose, maltose, D-mannose, 1, 6-dehydrated-beta-D-glucose, trehalose, methyl beta-D-galactopyranoside, D-arabinose, D-galacturonic acid, L-rhamnose, D-xylulose and arabitol.

Preferably, the mass percentage of the sugar in the content of the medlar outer vesicle is 30-40%.

Plant extracellular vesicles generally possess biological effects similar to those of plants. As extracellular vesicles derived from matrimony vine we found by non-targeted metabolome sequencing that the content of GqDNV was predominantly polysaccharides and lipids.

Preferably, the content of the medlar extracellular vesicles further comprises lipids, terpenes, nucleotides and derivatives thereof, organic acids, phenolic acids, alkaloids, quinones, lactones, amino acids and derivatives thereof, flavones, stilbenes, vitamins, lignans and coumarins.

Preferably, the content of the medlar extracellular vesicles comprises the following components in percentage by weight: 35.47% sugar, 33.05% lipid, 8.02% terpenes, 6.99% nucleotides and derivatives thereof, 6.27% organic acids, 5.06% phenolic acids, 1.69% alkaloids, 1.06% quinones, 0.70% lactones, 0.57% amino acids and derivatives thereof, 0.56% flavones, 0.24% stilbenes, 0.20% vitamins, 0.07% lignans and 0.05% coumarins.

In a second aspect, the invention provides a method for preparing the medlar extracellular vesicles, comprising the following steps:

(1) Grinding fresh Chinese wolfberry to obtain a Chinese wolfberry stock solution;

(2) Centrifuging the stock solution of the Chinese wolfberry, removing sediment, and taking supernatant to obtain crude suspension containing extracellular vesicles of the Chinese wolfberry;

(3) Centrifuging the crude suspension containing extracellular vesicles of fructus Lycii by using an ultracentrifuge, discarding supernatant, and collecting precipitate;

(4) Re-suspending and precipitating with phosphate buffer, adding to the uppermost layer of sucrose density gradient solution, and centrifuging; taking the interlayer solution with middle density to extract the extracellular vesicles of the medlar in the specific density area;

(5) Diluting the solution with PBS, centrifuging again, collecting precipitate, re-suspending the precipitate with PBS, filtering with a filter membrane, and removing residual non-vesicle large-particle-size particles to obtain extracellular vesicles of fructus Lycii.

Preferably, steps (2) - ((5) are all carried out at 4 ℃.

Preferably, the centrifugal treatment in the step (2) is carried out, wherein the centrifugal force and the centrifugal time are 1000g for 10min and 2000g for 20min in sequence; centrifuge 10000g for 60min.

Preferably, the centrifugation in step (3) is performed at 150,000g for 90min.

Preferably, the sucrose density gradient solution in the step (4) is a sucrose solution with different concentration gradients, and the concentration of the sucrose solution ranges from 8% to 60%; the gradient concentration of the sucrose is 8%,30%,45% and 60% from top to bottom respectively; the centrifugation is 150,000g for 90min.

Preferably, the middle density layer is a 30-45% density layer; the specific density region has a density of 1.13-1.19g/ml.

Preferably, the centrifugation in step (5) is a centrifugation at 150,000g for 90min.

Preferably, the filters obtained in step (5) are 0.44 μm and 0.22 μm filters, respectively, to exclude residual non-vesicle large size particles, resulting in a solution containing GqDNV.

The ultracentrifugation method is a gold standard for separating plant extracellular vesicles, and has the characteristics of low cost, low pollution rate and wide application range. In general, after the plants are broken up into liquids, they are centrifuged at a low speed (centrifugal force: 500-10,000 g) to remove dead cells and cell debris, and then centrifuged at a high speed (centrifugal force: 40,000-100,000 g) to enrich extracellular vesicles in the pellet. In practice, specific centrifugation conditions and times are often set according to the nature of the sample. Due to differences in plant composition and structure, extracellular vesicles obtained from different sources using the same isolation method may have large differences, reflected in particle size, yield or purity.

In density gradient centrifugation, particles having a higher density than the solvent will precipitate, while particles having a lower density will float. Thus, translucent bands appear between 8-30% density layer, 30-45% density layer and 45-60% density layer after centrifugation of the suspension. The 30-45% density interlayer solution was taken to extract extracellular vesicles specific for its characteristic density region (1.13-1.19 g/ml).

Compared with the existing common plant extracellular vesicle kit (coprecipitation method) and ultrafiltration method in the market, the method for combining the ultracentrifugation and the density gradient separation has the advantages of low cost, small pollution risk and large sample capacity. At present, no method for successfully extracting extracellular vesicles from medlar exists.

Preferably, the particle size of the medlar extracellular vesicles is 113.4-124.6nm.

Preferably, 1×10 fructus Lycii per 1kg can be extracted ¹² And (3) the medlar extracellular vesicles.

In a third aspect, the invention provides a pharmaceutical formulation comprising an extracellular vesicle of Lycium barbarum and a pharmaceutically acceptable carrier.

Preferably, the pharmaceutical preparation is administered by injection, gastric lavage or oral administration.

Preferably, the injection amount is 7.5 mg/kg.bw.

The invention has the beneficial effects that:

(1) By combining the ultracentrifugation method and the sucrose density gradient centrifugation method, the GqDNV is successfully separated from the fresh Chinese wolfberry, and the method for separating the GqDNV from the fresh Chinese wolfberry can be summarized stably and efficiently.

(2) The GqDNV is proved to be rich in bioactive substances such as medlar polysaccharide and the like.

(3) The application of the GqDNV is provided, for example, the GqDNV is used as an intervention object so as to achieve the effects of promoting tissue repair/growth and the like, and the GqDNV is used as a drug carrier to be applied to the medical field and the like. It has been demonstrated to improve motor function in mice with reduced muscle wilt and promote skeletal muscle growth. Although related researches are still in progress, the GqDNV has better bioavailability while having various biological active substances, can be directly absorbed by tissue cells in a intramuscular injection mode and the like, and has good medical application prospect.

Drawings

Fig. 1 is a flow chart of GqDNV extraction in an embodiment.

FIG. 2 is a GqDNV extraction experimental procedure; a-B: obtaining a medlar stock solution, C-F: ultracentrifugation is performed on the stock solution of medlar to remove impurities, G-K: and obtaining the GqDNV by density gradient centrifugation.

FIG. 3 is a GqDNV nanoparticle size analysis; the average grain diameter and concentration of the GqDNV, and the density and grain diameter distribution of the GqDNV.

Fig. 4 is a transmission microscope view of GqDNV.

Fig. 5 is GqDNV non-targeted metabolome results.

FIG. 6 shows GqDNV polysaccharide-targeted metabolome results.

FIG. 7 is a statistical plot of the grip of the mice (grip results are normalized by dividing by body weight); a: muscle wasting mice (control group) grip, b: muscle wasting mice (experimental group) with GqDNV dry prognosis.

Fig. 8 is the quadriceps (left side) of the mouse thigh; a: muscle-wasting mice (control group) quadriceps femoris, b: muscle wasting following GqDNV dry mice (experimental group) quadriceps femoris.

Fig. 9 is a cross-sectional HE stained section of mouse skeletal muscle (quadriceps femoris (left side)) myofiber; a: muscle wilt mice (control group) skeletal muscle fiber cross section, b: muscle wasting following GqDNV dry mice (experimental group) skeletal muscle fiber cross section.

Fig. 10 is a comparison of the cross-sectional areas of skeletal (quadriceps) muscle fibers of mice.

Detailed Description

For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the following specific examples.

Example 1: extraction method of extracellular vesicles GqDNV of Chinese wolfberry

1. The experimental method comprises the following steps:

taking fresh fructus Lycii, gently washing with double distilled water for three times, washing off residual contaminants (such as soil, etc.) on the surface, and grinding in a wall breaking machine for 1min to obtain fructus Lycii stock solution (figure 1, step A). Carrying out a subsequent series of centrifugal treatment at 4 ℃, separating and discarding large particles such as dead cell fragments of plants by utilizing the separation principle that particles with different particle diameters and densities have different sedimentation rates under the action of centrifugal force, and obtaining suspension containing GqDNV (figure 1, step B): centrifuging for 10min under the condition of 1000g centrifugal force, discarding precipitate, and collecting supernatant; centrifuging for 20min under the condition of 2000g centrifugal force, discarding precipitate, and collecting supernatant; centrifuging under 10000g centrifugal force for 60min, discarding precipitate, and collecting supernatant.

The suspension containing extracellular vesicles of Lycium barbarum collected in the previous step was centrifuged at 150,000g for 90min at 4℃using an ultracentrifuge (FIG. 1, step C), the supernatant was discarded, and the pellet (containing GqDNV) was taken. After the pellet was resuspended in phosphate buffer (Phosphate Buffer Saline, PBS), the suspension was gently added to the top layer of the prepared density gradient sucrose solution (8%, 30%,45%,60% from top to bottom respectively) and centrifuged at 150,000g for 90min at 4 ℃ (fig. 1, step D). In density gradient centrifugation, particles having a higher density than the solvent will precipitate, while particles having a lower density will float. Thus, translucent bands appear between 8-30% density layer, 30-45% density layer and 45-60% density layer after centrifugation of the suspension. The 30-45% density interlayer solution (FIG. 1, step E) was taken to extract extracellular vesicles specific for its characteristic density region (1.13-1.19 g/ml). The solution was diluted with PBS and centrifuged again at 150,000g for 90min at 4℃to obtain a pellet (GqDNV). After resuspension with PBS, the solution containing GqDNV was obtained by sequentially passing through 0.44 μm and 0.22 μm filters, and removing the residual non-vesicle large particle size particles. And (3) carrying out nano particle size analysis and transmission electron microscope observation on the obtained solution containing the GqDNV.

The density gradient sucrose solution configuration scheme is as follows: take 250ml of 8% sucrose solution as an example: 20g of sucrose, 20mmol/L of Tris-HCl (pH 7.2), 1mmol/L of ethylenediamine tetraacetic acid, dissolved in double distilled water, fixed in a 250ml volumetric flask, autoclaved and stored. 30%,45%,60% sucrose solution 250ml contained 75g, 112.5g and 150g sucrose respectively, and the rest of the components were identical to those of 8% sucrose solution according to the preparation method. Sucrose solutions were added to the centrifuge tube in order of 8%,30%,45%,60% sucrose solution density from high to low. During the preparation of the gradient sucrose solution, the sucrose solution needs to be slowly added, and the condition that the density gradient layers are mutually mixed is avoided.

2. Experimental results:

according to the invention, the GqDNV is extracted from fresh medlar for the first time, the feasibility of separating the GqDNV by an ultracentrifugation method is verified, and the GqDNV suspension obtained by successful separation is verified through nanoparticle tracking analysis and a transmission electron microscope, and the specific experimental result is shown in figure 2. Compared with other schemes for separating plant extracellular vesicles, the technical scheme is suitable for prolonging the low-speed centrifugation (the centrifugal force is 10,000g or below) time aiming at the characteristics of higher polysaccharide content and easy formation of colloidal substances in medlar; for particles that may be incorporated into the final isolated product extracellular vesicles, the other particles that may be incorporated are discarded in the final step by passing through the filter twice (0.44 μm and 0.22 μm filters).

In practical operation, we can extract 1×10 from 1kg fresh fructus Lycii by ultracentrifugation method and sucrose density gradient centrifugation method ¹² The nano particle size analysis of the GqDNV shows that the particle size of the extracted GqDNV is mainly concentrated in a 113.4-124.6nm section (figure 3) and is in a cup-disk shape under an electron transmission microscope (figure 4). Compared with the existing common plant extracellular vesicle kit (coprecipitation method) and ultrafiltration method in the market, the method for combining the ultracentrifugation and the density gradient separation has the advantages of low cost, small pollution risk and large sample capacity. Plant extracellular vesicles generally possess biological effects similar to those of plants. As extracellular vesicles derived from Lycium barbarum we found by non-targeted metabolome sequencing (FIG. 5), the content of GqDNV contained 35.47% saccharide, 33.05% lipid, 8.02% terpenes, 6.99% nucleotide and its derivativesBiological, 6.27% organic acid, 5.06% phenolic acid, 1.69% alkaloid, 1.06% quinone, 0.70% lactone compound, 0.57% amino acid and derivatives thereof, 0.56% flavone, 0.24% stilbene, 0.20% vitamin, 0.07% lignan, and 0.05% coumarin. It can be seen that the extracellular vesicles of Lycium barbarum are mainly composed of saccharides and lipids.

The invention further analyzes the components of the saccharides, and discovers that the compositions of the saccharides are as follows: sucrose, glucose, D-fructose, D-cellobiose, maltose, D-mannose, 1, 6-anhydro-beta-D-glucose, trehalose, methyl beta-D-galactopyranoside, D-arabinose, D-galacturonic acid, L-rhamnose, D-xylulose, arabitol. Among them, the higher content of several sugars are D-cellobiose, maltose, glucose, D-fructose and sucrose. D-cellobiose and maltose are both 0.0004mg/mL-0.0005mg/mL; the content of glucose and D-fructose is equivalent to about 0.5 mg/mL; sucrose content is as high as 1.5mg/mL.

Example 2: biological effects of GqDNV

1. The experimental method comprises the following steps:

25C 57BL6/J mice were intraperitoneally injected with dexamethasone solution (25 mg/kg. Bw) for 9 days to construct a muscle atrophy model, and after successful molding, a muscle atrophy state was maintained by daily intraperitoneal injection of dexamethasone solution (5 mg/kg. Bw). 25 mice successfully molded were randomly divided into 2 groups.

(1) Grouping

Experimental group: lycium barbarum extracellular vesicles+modeling (muscle atrophy) mice: taking 13C 57BL6/J mice with muscular atrophy modeling, and injecting 7.5 mg/kg.bw of medlar extracellular vesicle solution into the mice for 3 weeks once a day;

control group: PBS + model (muscle wasting) mice: 12 muscle atrophy-modeled C57BL6/J mice were given an equal volume of PBS by intramuscular injection to the extracellular vesicle solution of Lycium barbarum, once daily for 3 weeks.

(2) Grip test

The muscle strength of the mice before and after the intervention was measured using a mouse grimeter. The grip test adaptation training was performed daily on the first two days, and the formal grip test was performed on the third day. When the grabbing force is detected, the grabbing force meter is calibrated by the electronic scale, the mouse is calmed and horizontally placed on the grabbing force plate, and the tail of the mouse is pulled to the forelimbs of the mouse to loosen the grabbing force plate. The holding power is detected three times a day, the mice rest for at least 30min after each detection is finished, the next detection is carried out, and the maximum value of the holding power of the mice for 3 times is defined as the holding power of the mice.

(3) Observing the morphology of muscle fiber and counting the cross-sectional area

Each group of mice was H & E stained by taking the same portion of quadriceps femoris, and cutting out a tissue mass of 0.5 cm. Times.0.5 cm. Photographs were observed under an optical microscope, the morphological characteristics of the muscle fibers of the quadriceps femoris of each group of mice were observed, and the cross-sectional areas of the muscle fibers were calculated by Image J software.

2. Experimental results:

we found in animal experiments that skeletal muscle motor function of mice with muscle wasting C57BL/6 was also improved by intramuscular injection of GqDNV, and grip strength of mice with muscle wasting was significantly improved (FIG. 7). At the same time, skeletal muscle fiber cross-sectional area increased significantly after intramuscular injection of GqDNV in muscle-contracting mice (fig. 8-10), suggesting that GqDNV is able to promote skeletal muscle growth, but further effects and mechanisms thereof are still under investigation. The above results indicate that we can successfully extract GqDNV, and that GqDNV contains bioactive substances related to promoting skeletal muscle growth.

In conclusion, the extracellular vesicles from medlar are rich in polysaccharide substances. In the detection of the targeted metabolome, it was found that the saccharide of GqDNV is the main component. We have found in laboratory studies that GqDNV has the effect of improving muscle function in skeletal muscle of mice, reducing muscle function, and promoting skeletal muscle growth, but further mechanisms of action are under investigation. The results show that the GqDNV not only has various bioactive components, but also has high bioavailability, can improve the absorption and utilization degree of tissues by intramuscular injection and other modes, and has good medical application prospect. Meanwhile, the GqDNV serving as the extracellular vesicle of plant source has the advantages of wide source and high extraction rate, is easy to be absorbed by cells, and has potential as a drug carrier.

Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.

Claims (10)

1. Use of an extracellular vesicle of lycium barbarum, the contents of which comprise carbohydrates, in the manufacture of a medicament for promoting tissue repair or growth.

2. The use of claim 1, wherein the tissue is skeletal muscle.

3. The use according to claim 1, wherein the saccharide comprises at least one of sucrose, glucose, D-fructose, D-cellobiose, maltose, D-mannose, 1, 6-anhydro- β -D-glucose, trehalose, methyl β -D-galactopyranoside, D-arabinose, D-galacturonic acid, L-rhamnose, D-xylulose and arabitol.

4. Use according to claim 1 or 3, wherein the mass percentage of the saccharide in the content of the outer vesicles of wolfberry is 30-40%.

5. The use according to any one of claims 1 to 4, wherein the content of said extracellular vesicles of lycium barbarum further comprises lipids, terpenes, nucleotides and derivatives thereof, organic acids, phenolic acids, alkaloids, quinones, lactones, amino acids and derivatives thereof, flavones, stilbenes, vitamins, lignans and coumarins.

6. The use of claim 5, wherein the contents of the extracellular vesicles of lycium barbarum comprise the following components in weight percent: 35.47% sugar, 33.05% lipid, 8.02% terpenes, 6.99% nucleotides and derivatives thereof, 6.27% organic acids, 5.06% phenolic acids, 1.69% alkaloids, 1.06% quinones, 0.70% lactones, 0.57% amino acids and derivatives thereof, 0.56% flavones, 0.24% stilbenes, 0.20% vitamins, 0.07% lignans and 0.05% coumarins.

7. The use according to any one of claims 1 to 6, wherein the method for the preparation of extracellular vesicles of lycium barbarum comprises the steps of:

(1) Grinding fresh Chinese wolfberry to obtain a Chinese wolfberry stock solution;

(2) Centrifuging the stock solution of the Chinese wolfberry, removing sediment, and taking supernatant to obtain crude suspension containing extracellular vesicles of the Chinese wolfberry;

(3) Centrifuging the crude suspension containing extracellular vesicles of fructus Lycii by using an ultracentrifuge, discarding supernatant, and collecting precipitate;

(4) Re-suspending and precipitating with phosphate buffer, adding to the uppermost layer of sucrose density gradient solution, and centrifuging; taking the interlayer solution with middle density to extract the extracellular vesicles of the medlar in the specific density area;

(5) Diluting the solution with PBS, centrifuging again, collecting precipitate, re-suspending the precipitate with PBS, filtering with a filter membrane, and removing residual non-vesicle large-particle-size particles to obtain extracellular vesicles of fructus Lycii.

8. The use according to claim 1, wherein the extracellular vesicles of wolfberry have a particle size of 113.4-124.6nm.

9. A pharmaceutical formulation comprising an extracellular vesicle of lycium barbarum and a pharmaceutically acceptable carrier.

10. The pharmaceutical formulation of claim 9, wherein the pharmaceutical formulation is administered by injection, lavage or oral administration.