Preparation method and application of clematidoside exosome for preventing and treating alcoholic liver injury

Abstract

The invention discloses a preparation method and application of a clematidoside exosome for preventing and treating alcoholic liver injury. A clematidoside single component and a clematidoside-loaded ginger-sourced exosome composition can effectively reverse adverse reactions such as hepatomegaly, liver fat accumulation and the like caused by the alcoholic liver injury; the level contents of ALT, AST and ALP are reduced, and the possibility of resisting the alcoholic liver injury is realized. The fresh ginger-sourced exosome extracted and separated from fresh ginger has a double-lipid-layer structure, and the clematidoside is wrapped in the exosome, so that the active structure of the clematidoside is protected from being damaged, and the bioavailability of the clematidoside is improved. The fresh ginger-derived exosome loaded with clematidoside can improve the drug uptake ability of the liver, so that the treatment effect on the alcoholic liver injury is further improved.

Description

Technical Field

[0001] The present invention relates to a new use of a medicine and a preparation thereof, and in particular to a method for preparing lingxianxin glycoside exosomes for preventing and treating alcoholic liver damage and an application thereof. Background Art

[0002] Alcohol-induced liver damage, characterized by inflammation, oxidative stress, and cell damage within the liver, has caused significant health problems worldwide. The consequences of chronic alcohol consumption span a spectrum of liver diseases, including alcohol-related liver disease (ALD), ranging from simple steatosis (fatty liver) to more severe conditions such as alcoholic hepatitis and cirrhosis.

[0003] Clematichinenoside AR (AR) is a triterpenoid saponin, an active ingredient extracted from the root of the traditional Chinese herbal medicine Clematis chinensis. Clematis chinensis has a long history of treating diseases, but it is mainly used to treat limb pain caused by rheumatism and athlete's foot pain. It has the effect of removing rheumatism and unblocking meridians. It is used for rheumatic pain, limb numbness, tendon spasm, and poor flexion and extension (Pharmacopoeia of the People's Republic of China 2020 edition).

[0004] Exosomes are a type of extracellular vesicles secreted by cells with a diameter of 30 to 200 nm. They are usually involved in intercellular communication, regulating immune responses, etc. They can stably deliver their contents to target cells and avoid degradation. The main advantage of exosomes as a delivery system is that they are different from carriers in terms of excellent biocompatibility, low immunogenicity, long half-life and strong cellular uptake ability. Using exosomes as an in vivo delivery system for natural compounds is conducive to solving the problem of low in vivo bioavailability of natural compounds.

[0005] Although a large number of studies have demonstrated the various pharmacological effects of AR, there are no reports on the use of AR in the treatment of alcoholic liver damage (ALD). In addition, pharmacokinetics show that AR has low bioavailability, which greatly limits its clinical application. Therefore, it is necessary to modify the delivery system of AR to improve its bioavailability. Summary of the invention

[0006] Purpose of the invention: The purpose of the present invention is to provide linaloside which can be administered orally to treat alcohol-induced diseases in animals. The present invention also relates to a method for extracting and purifying linaloside from Clematis chinensis and encapsulating it in ginger exosomes to achieve the purpose of improving the efficacy of the drug in treating alcoholic liver damage.

[0007] Technical solution: ginger exosomes loaded with lingxianxinoside are prepared by mixing ginger exosomes and lingxianxinoside and then subjecting them to ultrasonic treatment. After the ultrasonic treatment, the mixture is incubated to restore the exosome membrane and then centrifuged to obtain the obtained product.

[0008] The ginger exosomes loaded with lingxianxinoside are ultrasonically treated in an environment of 0 to 4°C, and the ultrasonic conditions are: 10% to 30% amplitude, 4 to 8 cycles, 4 to 6s on, 2 to 3s off, and a pause of 1 to 4 minutes in the middle of each cycle; the incubation temperature is 30 to 40°C, and the time is 40 to 80 minutes.

[0009] The ginger exosomes loaded with lingxianxinoside are centrifuged at ultra-high speed at 120,000-150,000 g, the supernatant is discarded, and the precipitate is resuspended in a buffer solution.

[0010] The ginger exosomes loaded with lingxianxinoside have a concentration of 0.1-2.0 mg / mL of ginger exosomes, a concentration of lingxianxinoside of 0.1-2.0 mg / mL, and a mass ratio of ginger exosomes to lingxianxinoside of 1:6-6:1.

[0011] The ginger exosomes loaded with lingxianxinosidase are prepared by the following steps:

[0012] Take ginger, add buffer, crush and filter, and take the filtrate; first centrifuge the ginger juice at 800-1000g, then centrifuge the supernatant at 2000-3000g, then centrifuge the supernatant at 14000-15000g, and finally ultracentrifuge the supernatant at 120000-150000g, the above centrifugation temperature is all at 0-4°C, take the precipitate, and obtain the crude exosome; mix the above crude ginger exosome with PEG solution, incubate at 0-4°C overnight, take the incubated sample and centrifuge it at 10000-15000g, take the precipitate, and obtain ginger exosome.

[0013] The ginger exosomes loaded with lingxianxinoside, the PEG solution is a PEG-6000 solution with a mass percentage of 5-10%.

[0014] Application of lingxianxinoglucoside in preparing medicine for preventing or treating alcoholic liver damage.

[0015] Application of ginger exosomes loaded with chelidonin in the preparation of drugs for preventing or treating alcoholic liver damage.

[0016] In the application, the CAS number of lingxianxinoglucoside is 761425-93-8.

[0017] Application of ginger exosomes in the preparation of drugs for preventing or treating alcoholic liver damage.

[0018] In the treatment of alcoholic liver damage and diseases caused by it, the combination of ginger-derived exosomes and lingxianxinoside or the single component of lingxianxinoside is mixed with pharmaceutically acceptable pharmaceutical excipients using conventional preparation processes to form ointments, liniments, patches, lotions, solutions, powders, sprays, syrups, suppositories, tablets, pills, granules, capsules, injections or gastrointestinal sustained-release preparations, etc.

[0019] A method for preparing lingxianxin glycosides comprises the following steps:

[0020] Take the medicinal material of Clematis chinensis, grind it, add 6 to 18 times of 30% to 60% ethanol, soak it for 0.5 to 5 hours, heat it, and then reflux it for 2 to 4 times, each time for 1 to 4 hours; combine the filtrate, reduce pressure and concentrate it, add it into a two-phase aqueous extraction system, the mass fraction of ammonium sulfate is 16% to 24%, the mass fraction of ethanol is 26% to 32%, shake or stir it for 3 to 10 minutes, let it stand for 20 to 40 minutes to separate the phases, and extract it continuously for 4 to 6 times; take the upper phase solution, dry it under reduced pressure, add ethanol to desalt it, filter it, concentrate it and dry it, and obtain the crude product of clematis glycosides; take the crude product of clematis glycosides, dissolve it in 25 to 35% ethanol, filter it, prepare the sample by HPLC, and the chromatographic column filler is C 18 The column was prepared by using acetonitrile-water solution as the mobile phase and the ultraviolet detection wavelength was 180-220 nm. The target component solution was collected and dried to obtain the pure product of lingxianxinoglucoside.

[0021] Take fresh ginger tissue, add appropriate amount of PBS, crush and filter, and take the filtrate; first centrifuge the ginger juice at 800-1000g at 4°C, then centrifuge the supernatant at 2000-3000g, then centrifuge the supernatant at 14000-15000g, and finally ultracentrifuge the supernatant at 120000-150000g, all of which are centrifuged at 4°C, and take the precipitate to obtain crude exosomes. Mix the crude ginger exosomes with an equal volume of 8% PEG-6000 solution and incubate at 4°C overnight, take the incubated sample and centrifuge it at 10000-15000g, take the precipitate, and obtain ginger exosomes.

[0022] The ginger exosomes were mixed with lingxianxinoside and then ultrasonically treated. After the ultrasonic treatment, the exosome membrane was incubated to restore the exosome membrane, and the ginger exosomes loaded with lingxianxinoside were obtained by centrifugation. The concentration of ginger exosomes was 0.1-2.0 mg / mL, the concentration of lingxianxinoside was 0.1-2.0 mg / mL, and the mass ratio of ginger exosomes to lingxianxinoside was 1:6-6:1. The ultrasonic conditions were: 10%-30% amplitude, 4-8 cycles, 4-6s on, 2-3s off, and a 2-min pause in the middle of each cycle. During the ultrasonic process, the ginger exosomes and lingxianxinoside mixture were placed in an environment of 0-4°C, the incubation temperature was 30-40°C, and the time was 40-80min. After the incubation, the sample was ultra-centrifuged at 120000-150000g, the supernatant was discarded, and the precipitate was resuspended in a small amount of PBS.

[0023] Through animal experiments, an alcoholic liver injury model of zebrafish larvae was established. The experimental results showed that compared with the model group, the three different administration groups of lingxianxin, exosomes, and exosomes loaded with lingxianxin all improved liver enlargement and fat accumulation to a certain extent; the test results of AST, ALT, and ALP levels in zebrafish larvae also showed that compared with the model group, the administration groups were all downregulated to varying degrees, reducing the degree of liver cell damage in the body; the results of ROS level detection showed that compared with the model group, each administration group reduced the ROS level in zebrafish larvae and improved the inflammatory symptoms in the body. Moreover, the efficacy of exosomes loaded with lingxianxin is better than that of a single drug at the same dose, and has a significant anti-alcoholic liver injury effect.

[0024] The key technical points of the present invention are:

[0025] 1. A method for extracting and purifying clematisin from Clematis chinensis.

[0026] 2. Preparation and application of an exosome drug delivery system.

[0027] 3. Lingxianxin glycosides can treat alcoholic liver damage caused by alcohol.

[0028] 4. The combination of lindera glycosides encapsulated in ginger exosomes can treat alcoholic liver damage caused by alcohol, and the therapeutic effect is significantly higher than that of the same dose of lindera glycosides alone.

[0029] Beneficial effects: Compared with the prior art, the present invention has the following advantages: (1) The single component of lingxianxinoside and the ginger-derived exosome composition loaded with lingxianxinoside of the present invention can effectively reverse the adverse reactions such as liver enlargement and liver fat accumulation caused by alcoholic liver damage, reduce the levels of ALT, AST, and ALP, and have the potential to resist alcoholic liver damage. (2) The ginger-derived exosomes extracted and separated from ginger of the present invention have a double lipid layer structure, which encapsulates lingxianxinoside, protects its active structure from being destroyed, and improves the bioavailability of lingxianxinoside. (3) The ginger-derived exosomes loaded with lingxianxinoside of the present invention can improve the liver's ability to absorb drugs, thereby further improving the therapeutic effect on alcoholic liver damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the chromatogram of Lingxianxin glycosides;

[0031] Figure 2 Characterization diagram of exosomes loaded with lingxianxin glycosides: (A, B) exosome particle size; (C) exosome potential; (D) exosome SEM image. DETAILED DESCRIPTION

[0032] Example 1 Preparation and purification of Lingxianxin glycosides

[0033] Take 300g of Clematis chinensis, crush it and place it in a round-bottom flask, add 14 times the amount of 50% ethanol, soak for 1 hour, heat and reflux extraction 3 times, each time for 2.8 hours. Combine the filtrates, concentrate under reduced pressure and add a two-phase aqueous extraction system, with an ammonium sulfate mass fraction of 18% and an ethanol mass fraction of 28%, shake and stir for 5 minutes, let stand for 20 minutes, and extract continuously for 6 times. Take the upper phase and dry it under reduced pressure, add 20 times the amount of 95% ethanol, filter, take the filtrate, and dry it under reduced pressure to obtain the crude product of Clematis chinensis glycosides. Dissolve the crude product of Clematis chinensis glycosides in 30% methanol, filter, and prepare the sample by HPLC. The chromatographic column filler is C 18 Column, acetonitrile-water solution as mobile phase, UV detection wavelength of 203nm, collect the target component solution, dry, get 512mg of lingxianxin glycosides, purity of 95.53%, see Figure 1 .

[0034] Example 2 Preparation of ginger-derived exosomes loaded with lindera glycosides

[0035] Take 500 g of fresh ginger tissue, add an appropriate amount of PBS, crush and filter, and take the filtrate; centrifuge at 4°C, 800g for 30 minutes, and take the supernatant; as described above, complete centrifugation at a speed of 3000g and 15000g in sequence, discard the precipitate, and filter the supernatant through a 0.22 μm microporous filter membrane; place the filtrate in an ultra-high speed centrifuge tube, centrifuge at 4°C, 120000g, discard the supernatant, resuspend with 1×PBS, add an equal volume of 8% PEG-6000 solution, and incubate at 4°C overnight; centrifuge the incubated sample at 4°C, 10000g, and discard the supernatant; resuspend with 1×PBS, divide into portions, and store at -80°C. Ginger exosomes (1 mg / mL) were mixed with lingxianxin (2 mg / mL) and ultrasonicated in an ultrasonic disruptor; ultrasonic conditions: 20% amplitude, 6 cycles, 6s on, 4s off, 2min cooling between each cycle, and the mixture was placed on ice for the entire process. The mixture was centrifuged at 4000g for 15min in a 100KD ultrafiltration tube to remove free lingxianxin, and ginger-derived exosomes loaded with lingxianxin were obtained.

[0036] The characterization results of the exosomes derived from ginger loaded with lingxianxin glycosides are as follows Figure 2 As shown, A and B are the particle size distribution of exosomes loaded with lingxianxin glycosides detected by Malvern Nano Particle Size Analyzer, C is the Zeta potential of exosomes loaded with lingxianxin glycosides by Malvern Zeta Potential Analyzer, and D is the morphology of exosomes loaded with lingxianxin glycosides characterized by scanning electron microscopy (SEM). As can be seen from the figure, the particle size of exosomes loaded with lingxianxin glycosides is relatively uniform, concentrated at 208nm; it is electronegative, and the Zeta potential is concentrated at -14.45mV.

[0037] Example 3 Protective effect of single component of lingxianxinoside and ginger-derived exosome composition loaded with lingxianxinoside on the liver of zebrafish

[0038] Male and female zebrafish were raised separately in a 28°C circulating water system, maintained at 14h light and 10h dark per day, and fed Artemia twice. Three to four pairs (female: male = 1:1) of zebrafish around 6 months old were selected for mating and spawning, and normal embryos were collected into zebrafish embryo culture medium and placed in a 28°C constant temperature incubator to maintain the same lighting conditions for culture.

[0039] The zebrafish larvae in good condition at 5 days after fertilization were selected and transferred to a 6-well plate, with 30 fish per well and 1 well per group. The fish were randomly divided into the following groups: blank control group, model group, and drug group (single lindane glycosides drug group: lindane glycosides 15μM, lindane glycosides 30μM; ultrasound-loaded drug combination: ginger exosomes-lindane glycosides 15μM, ginger exosomes-lindane glycosides 30μM; simple mixed drug combination: ginger exosomes + lindane glycosides 30μM; single exosome group: ginger exosome group 200μg / mL). The model group and drug group were first soaked in 1.5% ethanol for 32h, and then the model group was soaked in blank culture medium and the drug group was soaked in the culture medium of the corresponding drug for 48h.

[0040] After modeling and drug administration, each group of zebrafish was anesthetized with a solution containing 2.5 g / L tricaine, and the stained images of zebrafish in prone position were taken using a stereomicroscope (olympuszx16), and the liver area and grayscale value were calculated using Image J software. All data are presented in the form of mean ± standard deviation (SD). The significance of the difference was determined by one-way analysis of variance (ANOVA) using GraphPad Prism8.0.2 software.

[0041] As shown in the table below, in the experiment of liver morphology observation, compared with the model group, the liver size of zebrafish in different dosing groups improved to varying degrees, the drug combination encapsulated by exosomes was better than a single drug at the same dose, and the drug combination constructed by ultrasound-assisted drug delivery system was better than the simple mixed drug combination.

[0042] Table 1 Effects of different drug administration groups on zebrafish liver morphology (n=10, )

[0043]

[0044] Note: Compared with the control group, ### P<0.001; compared with the model group, * P<0.05, ** P<0.01, *** P<0.001.

[0045] Example 4 Effects of single component of linaloside and linaloside-loaded ginger-derived exosome composition on lipid accumulation in zebrafish liver

[0046] Male and female zebrafish were raised separately in a 28°C circulating water system, maintained at 14h light and 10h dark per day, and fed Artemia twice. Three to four pairs (female: male = 1:1) of zebrafish around 6 months old were selected for mating and spawning, and normal embryos were collected into zebrafish embryo culture medium and placed in a 28°C constant temperature incubator to maintain the same lighting conditions for culture.

[0047] The zebrafish larvae in good condition at 5 days after fertilization were selected and transferred to a 6-well plate, with 30 fish per well and 1 well per group. The animals were randomly divided into the following groups: blank control group, model group, and drug-treated group (15 μM lingxianxinoside, 30 μM lingxianxinoside, 15 μM ginger exosome-lingxianxinoside, 30 μM ginger exosome-lingxianxinoside, and ginger exosome group). The model group and drug-treated group were first soaked in 1.5% ethanol for 32 hours, and then the model group was soaked in blank culture medium and the drug-treated group was soaked in the culture medium of the corresponding drug for 48 hours.

[0048] After modeling and drug administration, the liver of zebrafish samples in each group was stained with Oil Red O. The specific steps are as follows:

[0049] (1) Rinsing: Each group of zebrafish larvae were washed three times with embryo culture medium, each time in a shaker at 4°C for 15 min to fully wash away the modeling agent or drug solution;

[0050] (2) Fixation: The thoroughly washed zebrafish larvae of each group were fixed with 4% paraformaldehyde fixative at 4°C overnight;

[0051] (3) Rinsing: Wash each group of fixed zebrafish again with embryo culture medium until there is no smell of paraformaldehyde, shaking for about 10 minutes each time;

[0052] (4) Staining: Mix the stock solution of Oil Red O and pure water in a ratio of 3:2, and filter through a 0.22 μm microporous filter membrane. Then stain the rinsed zebrafish sample with the prepared 0.5% Oil Red O solution in the dark for 1 hour;

[0053] (5) Decolorization: Remove the staining solution and add 60% isopropanol for decolorization twice, each time for 2 to 3 minutes;

[0054] (6) Rinse: After decolorization, wash thoroughly with PBS for 3 times, 15 minutes each time, until there is no mutual repulsion between the fish. After thorough washing, store in 70% glycerol at 4°C.

[0055] Finally, the zebrafish larvae were placed and fixed on a slide containing 50% glycerol, and the staining images of zebrafish in prone position were taken using a stereo microscope (olympuszx16), and the oil red staining area and grayscale value were calculated using image J software. All data are presented in the form of mean ± standard deviation (SD). The significance of the difference was determined by one-way analysis of variance (ANOVA) using GraphPad Prism 8.0.2 software.

[0056] As shown in the table below, in the oil red staining observation experiment, compared with the model group, the grayscale value of the zebrafish liver in different dosing groups was significantly reduced, the drug combination encapsulated by exosomes was better than the single drug at the same dose, and the drug combination constructed by ultrasound-assisted drug delivery system was better than the simple mixed drug combination.

[0057] Table 2 Effects of different drugs on zebrafish oil red staining (n=10, )

[0058]

[0059]

[0060] Note: Compared with the control group, ### P<0.001; compared with the model group, * P<0.05, ** P<0.01, *** P<0.001.

[0061] Example 5 Effects of single component of linaloside and linaloside-loaded ginger-derived exosome composition on ROS levels in zebrafish

[0062] Male and female zebrafish were raised separately in a 28℃ circulating water system, maintained with 14h light and 10h dark per day, and fed with Artemia twice. Three to four pairs (female: male = 1:1) of zebrafish around 6 months old were selected for mating and spawning, and normal embryos were collected into zebrafish embryo culture medium and placed in a 28℃ constant temperature incubator to maintain the same lighting conditions for culture.

[0063] The zebrafish larvae in good condition at 5 days after fertilization were selected and transferred to a 6-well plate, with 30 fish per well and 1 well per group. The fish were randomly divided into the following groups: blank control group, model group, and drug-treated group (15 μM lingxianxinoside, 30 μM lingxianxinoside, ginger exosome-lingxianxinoside 1 5 μM, ginger exosome-lingxianxinoside 30 μM, ginger exosome group). The model group and drug-treated group were first soaked in 1.5% ethanol for 32 hours, and then the model group was soaked in blank culture medium and the drug-treated group was soaked in the culture medium of the corresponding drug for 48 hours.

[0064] After modeling and drug administration, zebrafish were stained with a final concentration of 10 μmol / L DCFH-DA fluorescent probe at room temperature in the dark for 1 h, washed three times with PBS, observed under a fluorescence microscope and photographed, and the average fluorescence intensity was calculated using Image J software to indicate the level of ROS. All data are presented as mean ± standard deviation (SD). The significance of the difference was determined by one-way analysis of variance (ANOVA) using GraphPad Prism 8.0.2 software.

[0065] As shown in the table below, in the ROS level detection experiment, compared with the model group, the ROS levels of zebrafish in different dosing groups were reduced, the drug combination encapsulated by exosomes was better than a single drug at the same dose, and the drug combination using ultrasound to construct a drug delivery system was better than the drug combination with simple mixing.

[0066] Table 3 Effects of different drugs on ROS levels in zebrafish (n=10, )

[0067]

[0068]

[0069] Note: Compared with the control group, ### P<0.001; compared with the model group, * P<0.05, ** P<0.01, *** P<0.001.

[0070] Example 6 Effects of single component of linaloside and ginger-derived exosome composition loaded with linaloside on ALT, AST and ALP in zebrafish

[0071] Male and female zebrafish were raised separately in a 28°C circulating water system, maintained at 14h light and 10h dark per day, and fed Artemia twice. Three to four pairs (female: male = 1:1) of zebrafish around 6 months old were selected for mating and spawning, and normal embryos were collected into zebrafish embryo culture medium and placed in a 28°C constant temperature incubator to maintain the same lighting conditions for culture.

[0072] The zebrafish larvae in good condition at 5 days after fertilization were selected and transferred to a 6-well plate, with 30 fish per well and 1 well per group. The animals were randomly divided into the following groups: blank control group, model group, and drug-treated group (15 μM lingxianxinoside, 30 μM lingxianxinoside, 15 μM ginger exosome-lingxianxinoside, 30 μM ginger exosome-lingxianxinoside, and ginger exosome group). The model group and drug-treated group were first soaked in 1.5% ethanol for 32 hours, and then the model group was soaked in blank culture medium and the drug-treated group was soaked in the culture medium of the corresponding drug for 48 hours.

[0073] After modeling and drug administration of zebrafish according to 1.2, 30 zebrafish larvae specimens were collected from each group in a 1.5 mL Eppendorf tube, the drug solution was dried, and the weight was weighed. Precooled saline was added according to the weight-to-volume ratio of zebrafish and precooled saline = 1:9 (g / mL), the Eppendorf tube was placed on ice, and the tissue was homogenized (precooled) with a tissue grinder to fully lyse the tissue. After the tissue was fully lysed, it was centrifuged at 2500 rpm at 4°C for 10 minutes, and the supernatant was taken. ALT, AST, and ALP levels were determined according to the instructions of the kit. All data are presented as mean ± standard deviation (SD). The significance of the difference was determined by one-way analysis of variance (ANOVA) using GraphPad Prism 8.0.2 software.

[0074] As shown in the table below, compared with the blank group, the levels of ALT, AST, and ALP in the model group increased significantly, while different dosing groups showed different degrees of inhibitory effects on the levels of ALT, AST, and ALP. The drug combination encapsulated by exosomes was better than a single drug at the same dose, and the drug combination of the drug delivery system constructed by ultrasound was better than the drug combination of simple mixing.

[0075] Table 4 Effects of different drugs on the levels of ALT, AST and ALP in zebrafish (n=10, )

[0076]

[0077]

[0078] Note: Compared with the control group, ### P<0.001; compared with the model group, * P<0.05, ** P<0.01, *** P<0.001.

Claims

1. A ginger exosome loaded with lingxianxin glycoside, characterized in that: The ginger exosomes were mixed with lingxianxin and then subjected to ultrasonic treatment. After the ultrasonic treatment, the mixture was incubated to restore the exosome membrane and then centrifuged to obtain the product.

2. The ginger exosomes loaded with lingxianxinoside according to claim 1, characterized in that: Ultrasound is carried out in an environment of 0 to 4°C, and the ultrasonic conditions are: 10% to 30% amplitude, 4 to 8 cycles, 4 to 6 seconds on, 2 to 3 seconds off, and a pause of 1 to 4 minutes in the middle of each cycle; the incubation temperature is 30 to 40°C, and the time is 40 to 80 minutes.

3. The ginger exosomes loaded with lingxianxinoside according to claim 1, characterized in that: The centrifugation is to perform ultra-high speed centrifugation on the sample at 120,000-150,000 g, discard the supernatant, and resuspend the precipitate in a buffer solution.

4. The ginger exosomes loaded with lingxianxinoside according to claim 1, characterized in that: The concentration of ginger exosomes is 0.1-2.0 mg / mL, the concentration of lingxianxin is 0.1-2.0 mg / mL, and the mass ratio of ginger exosomes to lingxianxin is 1:6-6:

1.

5. The ginger exosomes loaded with lingxianxinoside according to claim 1, characterized in that: The ginger exosomes are prepared by the following steps: Take ginger, add buffer, crush and filter, and take the filtrate; first centrifuge the ginger juice at 800-1000g, then centrifuge the supernatant at 2000-3000g, then centrifuge the supernatant at 14000-15000g, and finally ultracentrifuge the supernatant at 120000-150000g, the above centrifugation temperature is all at 0-4°C, take the precipitate, and obtain the crude exosome; mix the above crude ginger exosome with PEG solution, incubate at 0-4°C overnight, take the incubated sample and centrifuge it at 10000-15000g, take the precipitate, and obtain ginger exosome.

6. The ginger exosomes loaded with lingxianxinoside according to claim 5, characterized in that: The PEG solution is a PEG-6000 solution with a mass percentage of 5 to 10%.

7. Use of Lingxianxin glycosides in the preparation of drugs for preventing or treating alcoholic liver damage.

8. Application of ginger exosomes loaded with chelidonin in the preparation of drugs for preventing or treating alcoholic liver damage.

9. The use according to claim 7 or 8, characterized in that: The CAS number of lingxianxinoside is 761425-93-8.

10. Application of ginger exosomes in the preparation of drugs for preventing or treating alcoholic liver damage.

Images