A nano vesicle from aloe vera containing quercetin and capable of protecting alcoholic liver injury and preparation and application thereof

Abstract

The application discloses a nano vesicle containing quercetin and capable of protecting alcoholic liver injury, and a preparation and application thereof. Quercetin is an active component with antioxidant and anti-inflammatory effects contained in gold potted orchid, however, its in-vivo curative effect is reduced due to its low water solubility and bioavailability, and its clinical curative effect is limited. The method for obtaining quercetin by separating gold potted orchid-derived nano vesicles has the advantages of simple operation, rapidness and the like, and the lipid bilayer of the exosome-like vesicle can maintain the stability of quercetin for a long time as a natural protective film of quercetin. When acting on HepG2 cells, the gold potted orchid-derived nano vesicle can reasonably regulate the liver index of mice, effectively prevent or inhibit adverse reactions such as hepatocyte edema, fatty degeneration and lipid droplet accumulation caused by alcohol intake of the model, and has a significant curative effect on alcoholic liver disease.

Description

Technical Field

[0001] This invention relates to the field of plant exosome-like vesicle isolation and extraction and anticancer drug research technology. More specifically, it relates to the extraction of Anoectochilus roxburghii-derived nanovesicles containing active drug components, and a feasibility study of the Anoectochilus roxburghii-derived nanovesicles as a liver-protective drug. Background Technology

[0002] Exosomes are nanoscale vesicles actively secreted by cells. They possess enhanced permeability and retention (EPR) effects and protect their contents. They also carry relevant genetic information from parent cells (such as proteins and nucleic acids), exhibiting excellent targeting capabilities to homologous cells. They can participate in intercellular communication and even cross the blood-brain barrier, bypassing the cellular P-glycoprotein drug efflux system, offering advantages for intracerebral drug delivery and reducing cellular drug resistance research. Furthermore, because exosomes contain a lipid bilayer, they effectively protect their internal active ingredients, ensuring they are not degraded before reaching the target site, and can serve as drug carriers for drug delivery. In recent years, with further understanding and exploration of exosomes, researchers have discovered their enormous potential in drug delivery and disease treatment.

[0003] However, exosomes currently used as drug carriers are mainly derived from human cells and human body fluids. These sources have low exosome content, high separation costs, and complex purification methods. Furthermore, tumor-derived exosomes may trigger tumor spread or immune responses, thus limiting their application in drug delivery research.

[0004] Plant-derived nanovesicles (PDNVs) are nanoscale vesicles secreted by plant cells. They share significant morphological and functional similarities with exosomes and offer a novel approach to addressing the aforementioned challenges. PDNVs are easy and inexpensive to isolate and extract; they carry various lipids, proteins, nucleic acids, and parental bioactive molecules that act synergistically on different pathways, demonstrating better therapeutic efficacy compared to single active ingredients extracted from plants. As exosome-like vesicles with a lipid bilayer structure, PDNVs' phospholipid structure acts as a natural protective barrier, preventing degradation or inactivation of their internal active ingredients and improving bioavailability. They exhibit low immunogenicity, high safety, and are made from natural components, exhibiting good biocompatibility in vivo. They can serve as a novel nano-drug delivery formulation for rapid and effective disease treatment. They also have good oral bioavailability, are stable in the gastrointestinal tract, and are not easily digested or degraded by gastric juice, pancreatic juice, or bile, thus maintaining their activity. Based on the above advantages, plant exosome-like vesicles have great potential for research and application in drug carriers.

[0005] *Anoectochilus roxburghii*, a perennial herbaceous plant belonging to the genus *Anoectochilus* in the Orchidaceae family, is a precious plant in traditional Chinese medicine, containing quercetin, an active ingredient with antioxidant and anti-inflammatory effects. Studies have shown that *Anoectochilus roxburghii* is effective in treating liver damage caused by carbon tetrachloride (CCl4), non-alcoholic fatty liver disease, autoimmune hepatitis, and chronic hepatitis B. However, oral administration of *Anoectochilus roxburghii* also presents some challenges, such as high cost, easy inactivation of active ingredients during processing, and poor bioavailability. Quercetin is an active ingredient in *Anoectochilus roxburghii* with antioxidant and anti-inflammatory effects. It can restore intracellular glutathione levels, reduce cellular oxidative stress, and decrease the formation of the lipid peroxide malondialdehyde. However, its low water solubility and bioavailability reduce its in vivo efficacy, limiting its clinical efficacy. These problems greatly restrict the pharmaceutical feasibility and clinical application of *Anoectochilus roxburghii* in oral dosage forms. The *Anoectochilus roxburghii*-derived nanovesicles possess a lipid bilayer structure similar to exosomes, serving as a natural protective membrane that reduces the impact of the external environment on their internal components. Furthermore, the presence of the lipid bilayer also increases the stability of quercetin to some extent. Summary of the Invention

[0006] The purpose of this invention is to provide a *Anoectochilus roxburghii*-derived nanovesicle containing quercetin that can protect against alcoholic liver injury, as well as its preparation method and application.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A type of *Anoectochilus roxburghii*-derived nanovesicle containing quercetin and exhibiting protective effects against alcoholic liver injury, along with its preparation method and applications, comprising the following steps:

[0009] S1: Preparation of *Anoectochilus roxburghii*-derived nanovesicles:

[0010] S1-1: Juice the fresh whole plant of Anoectochilus roxburghii using a juicer, filter it with gauze, and obtain the filtrate;

[0011] S1-2: Centrifuge the filtrate obtained in step S1-1 at high speed, discard the precipitate, and obtain the supernatant;

[0012] S1-3: The supernatant obtained in step S1-2 is subjected to ultracentrifugation, the precipitate is collected and resuspended in PBS to obtain a suspension of Anoectochilus roxburghii-derived nanovesicles containing quercetin and which can protect against alcoholic liver injury.

[0013] The juicing process requires mixing the fresh whole plant of *Anoectochilus roxburghii* with pre-cooled PBS or PBS ice cubes.

[0014] The process conditions for high-speed centrifugation are: centrifugation at 300–10000g for 10–60 min;

[0015] The process conditions for ultracentrifugation are: centrifugation at 10000g for 20 minutes;

[0016] S2: Pretreatment method for analysis of active ingredients in *Anoectochilus roxburghii*-derived nanovesicles:

[0017] S2-1: Centrifuge the suspension of Anoectochilus roxburghii-derived nanovesicles obtained in step S1, discard the supernatant, and obtain Anoectochilus roxburghii-derived nanovesicle precipitate.

[0018] S2-2: The *Anoectochilus roxburghii*-derived nanovesicle precipitate obtained in step S2-1 is resuspended in an appropriate amount of methanol to obtain a *Anoectochilus roxburghii*-derived nanovesicle resuspension, which is then fully vortexed and ultrasonically treated (500W, 15min).

[0019] S2-3: Centrifuge the resuspended liquid after sonication in step S2-2, and collect the supernatant and precipitate separately for later use;

[0020] S2-4: Resuspend the precipitate obtained in step S2-3 with an appropriate amount of methanol, vortex the resuspended liquid thoroughly and sonicate it (500W, 15min), centrifuge, collect the supernatant and combine it with the supernatant obtained in step S2-3.

[0021] S2-5: The resuspended solution obtained in step S2-4 was concentrated by nitrogen blowing to evaporate the solvent. After filtration, the Anoectochilus roxburghii-derived nanovesicles were obtained as a test sample and stored at 4°C for later use.

[0022] S3: Key parameters for high-performance liquid chromatography analysis of active ingredients in *Anoectochilus roxburghii*-derived nanovesicles:

[0023] The mobile phase conditions were methanol-0.3% phosphoric acid aqueous solution (volume ratio 55:45);

[0024] The column temperature is 30℃;

[0025] The detection wavelength is 375nm;

[0026] S4: Cytotoxicity study of quercetin and Anoectochilus roxburghii-derived nanovesicles:

[0027] S4-1: The cell suspension of human liver cancer cells HepG2 was evenly seeded into a 96-well plate. After the cells adhered, the culture medium was aspirated.

[0028] S4-2: Drug administration: Quercetin standard and Anoectochilus roxburghii-derived nanovesicles were added to different wells of a 96-well plate, incubated in a 37°C incubator for 24 hours, and then aspirated.

[0029] S4-3: Add MTT solution to a 96-well plate, react for 4 hours, and then remove the solution.

[0030] S4-4: Add dimethyl sulfoxide (DMSO) to a 96-well plate, shake, and measure the absorbance using an ELISA reader to calculate cell viability;

[0031] In step S4-1, the concentration of HepG2 cell suspension is 5000 cells / well, and the volume of cell suspension per well is 200 μL;

[0032] In step S4-2, the dosage concentrations of quercetin standard were 0.35 ng / mL and 0.70 ng / mL; the dosage concentrations of Anoectochilus roxburghii-derived nanovesicles were 25 ug / mL and 50 ug / mL (containing quercetin concentrations of 0.35 ng / mL and 0.70 ng / mL, respectively).

[0033] In step S4-3, the concentration of MTT is 5 mg / mL, and the volume added to each well is 20 μL;

[0034] In step S4-4, the volume of DMSO is 150 μL, and the detection wavelength is 570 nm.

[0035] S5: Verification of the effect of quercetin standard and Anoectochilus roxburghii-derived nanovesicles on the resistance of HepG2 cells to alcohol damage using the MTT assay:

[0036] S5-1: HepG2 cell suspension was inoculated into 96-well plates. After the cells adhered, the culture medium was aspirated and the cells were divided into drug administration group, alcohol group and blank control group.

[0037] S5-2: Administration: Quercetin standard and Anoectochilus roxburghii-derived nanovesicles were added to the administration groups, respectively, and incubated in a 37°C incubator for 24 hours before being aspirated; except for the blank control group, 5% alcohol was added to the other groups, and PBS was added to the blank control group, and the groups were incubated in a 37°C incubator for 24 hours before being aspirated.

[0038] S5-3: Add MTT solution to a 96-well plate, react for 4 hours, and then remove the solution.

[0039] S5-4: Add dimethyl sulfoxide (DMSO) to a 96-well plate, shake, and measure the absorbance using an ELISA reader to calculate cell viability;

[0040] In step S5-1, the cell suspension concentration is 5000 cells / well, and the cell suspension volume per well is 200 μL;

[0041] In step S5-2, the dosage concentrations of quercetin standard were 0.35 ng / mL and 0.70 ng / mL; the dosage concentrations of Anoectochilus roxburghii-derived nanovesicles were 25 ug / mL and 50 ug / mL (containing quercetin concentrations of 0.35 ng / mL and 0.70 ng / mL, respectively).

[0042] In step S5-3, the concentration of MTT is 5 mg / mL, and the volume added to each well is 20 μL;

[0043] In step S5-4, the volume of DMSO is 150 μL, and the detection wavelength is 570 nm;

[0044] S6: Immunohistochemical analysis of pathological changes in mouse livers before and after incubation with quercetin standards and *Anoectochilus roxburghii*-derived nanovesicles was used. Twenty-one healthy male ICR mice weighing 20-24g were randomly divided into a blank control group, an alcohol group, and an administration group. The administration group included ADNVs at doses of 50mg / kg and 25mg / kg (containing quercetin concentrations of 0.70μg / kg and 0.35μg / kg, respectively), corresponding doses of quercetin (0.70μg / kg and 0.35μg / kg), and a silymarin (50mg / kg) positive control group, with three mice in each group. After three days of acclimatization, the mice were administered the drugs via gavage once daily. The administration groups received high and low doses of ADNVs, corresponding concentrations of quercetin, and silymarin, respectively. The control and alcohol groups received an equal volume of PBS daily. An acute alcoholic liver injury model was established after 9 days. Two hours after administration on the ninth day, all mice except the blank control group received 50% ethanol (12 mL / kg) by gavage. After ethanol administration, mice were kept from food but allowed free access to water. They were weighed 12 hours later, and their livers were harvested for proportional pathological observation.

[0045] Mice were euthanized immediately after blood collection, and the livers were separated. After rinsing in physiological saline, the surface of the liver tissue was blotted dry with filter paper. Liver lesions were visually observed, and the liver weight was weighed and recorded. Finally, liver tissue sections were prepared and stained with hematoxylin-eosin (H&E) for observation. The steps for preparing pathological sections are as follows:

[0046] S6-1: Immerse the tissue in 4% paraformaldehyde for 24 hours for fixation. Remove the fixed material, trim it to make the cut surface smooth, and dehydrate and clear it. Then place the cleared tissue block in melted paraffin and keep it in a paraffin bath. After the paraffin has completely immersed the tissue block, embed and section it. Float the sections on 40℃ warm water to flatten them, then use anti-detachment slides to retrieve them, bake the slides, and store them.

[0047] S6-2: The paraffin in the sections was removed by xylene, then stained with hematoxylin-eosin solution, and then separated and dehydrated and cleared.

[0048] In step S6-1, the dehydration and clearing treatment sequence is as follows: dehydration overnight in 75% ethanol, dehydration for 2 hours in 85% ethanol, dehydration for 2 hours in 90% ethanol, dehydration for 1 hour in 95% ethanol, dehydration for 30 minutes in 100% ethanol I, and dehydration for 30 minutes in 100% ethanol II; then, the tissue is cleared in 1 / 2 anhydrous ethanol + 1 / 2 xylene for 5–10 minutes, cleared in xylene I for 5–10 minutes, and cleared in xylene II for 5–10 minutes; the section thickness is 4 μm.

[0049] In step S6-2, the specific process of dewaxing xylene is as follows: dewaxing in xylene I for 10 min; dewaxing in xylene II for 10 min; b. dewaxing in 100% ethanol I for 5 min; dewaxing in 100% ethanol II for 5 min; ethanol in 95% ethanol for 5 min; ethanol in 85% ethanol for 5 min; ethanol in 75% ethanol for 5 min; and then washing away the ethanol with distilled water for 5 min.

[0050] The significant advantages of this invention are:

[0051] (1) This invention relates to the extraction of exosome-like vesicles from Anoectochilus roxburghii containing quercetin active ingredient, which has a lipid bilayer that can protect the internal active ingredient and can effectively maintain the stability of the internal active ingredient.

[0052] (2) The *Anoectochilus roxburghii*-derived nanovesicles obtained in this invention can reasonably regulate the liver body index in mice, effectively prevent or inhibit adverse reactions such as hepatocyte edema, fatty degeneration, and lipid droplet accumulation caused by alcohol intake, and have significant therapeutic effects on alcoholic liver disease. Attached Figure Description

[0053] Figure 1 Figure 1 shows the characterization results of *Anoectochilus roxburghii*-derived nanovesicles. A, Particle size distribution of *Anoectochilus roxburghii*-derived nanovesicles detected by NTA; B, Zeta potential of *Anoectochilus roxburghii*-derived nanovesicles measured by Litesizer 500 nanometer particle size analyzer and Zeta potential analyzer; C, Morphology of *Anoectochilus roxburghii*-derived nanovesicles characterized by transmission electron microscopy (TEM).

[0054] Figure 2 Qualitative diagrams of quercetin standard and Anoectochilus roxburghii-derived nanovesicles.

[0055] Figure 3 This is a cytotoxicity diagram of quercetin standard and Anoectochilus roxburghii-derived nanovesicles.

[0056] Figure 4 The figure shows the effect of quercetin standard and Anoectochilus roxburghii-derived nanovesicles on alcohol-induced HepG2 cytotoxicity.

[0057] Figure 5These are H&E-stained pathological sections of liver tissue treated with quercetin standard and *Anoectochilus roxburghii*-derived nanovesicles. A, blank control group; B, alcohol group; C, 50 mg / kg silymarin group; D, 50 mg / kg ADNVs group; E, 0.70 μg / kg quercetin standard group; F, 25 mg / kg ADNVs group; G, 0.35 μg / kg quercetin standard group. Detailed Implementation

[0058] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0059] Example 1:

[0060] This embodiment provides a method for preparing *Anoectochilus roxburghii*-derived nanovesicles (ADNVs), the preparation steps of which are as follows:

[0061] (1) Juice the fresh whole plant of Anoectochilus roxburghii for 1 minute using a juicer and filter it through gauze to obtain the filtrate;

[0062] (2) The filtrate obtained in step (1) is centrifuged at high speed and the supernatant is obtained by removing the precipitate;

[0063] (3) After ultracentrifuging the supernatant obtained in step (3), the precipitate was collected and resuspended in an appropriate amount of PBS (0.1M, pH=7.2-7.4) to obtain a suspension of Anoectochilus roxburghii-derived nanovesicles;

[0064] In step (1), the juicing process requires mixing fresh whole herb of Anoectochilus roxburghii with PBS pre-cooled at 4°C or PBS ice cubes, and controlling the mass-volume ratio of fresh whole herb of Anoectochilus roxburghii to PBS to be 1:3.

[0065] In step (2), the conditions for high-speed centrifugation are: 4℃, 300~10000g centrifugation for 10~60min;

[0066] In step (3), the conditions for ultracentrifugation are: 4℃, 10000g centrifugation for 20min.

[0067] The characterization results of the obtained *Anoectochilus roxburghii*-derived nanovesicles are as follows: Figure 1As shown in the figure, A represents the particle size distribution of *Anoectochilus roxburghii*-derived nanovesicles detected by NTA; B represents the zeta potential of *Anoectochilus roxburghii*-derived nanovesicles obtained by Litesizer 500 nanoparticle size analysis and Zeta potential analysis; and C represents the morphology of *Anoectochilus roxburghii*-derived nanovesicles characterized by transmission electron microscopy (TEM). As can be seen from the figure, the *Anoectochilus roxburghii*-derived nanovesicles have a relatively uniform particle size, concentrated at 250 nm; they are electronegative, with a zeta potential concentrated at -7.93 mV; and they exhibit a complete saucer-like morphology with good dispersibility.

[0068] Example 2

[0069] The active components in the *Anoectochilus roxburghii*-derived nanovesicles prepared in Example 1 were analyzed using high-performance liquid chromatography (HPLC). The steps are as follows:

[0070] (1) Centrifuge the Anoectochilus roxburghii-derived nanovesicle suspension obtained in Example 1, discard the supernatant, and obtain the precipitate;

[0071] (2) The precipitate was resuspended in an appropriate amount of methanol to obtain a resuspended solution, which was then fully vortexed and ultrasonically treated.

[0072] (3) Centrifuge the resuspended solution after ultrasound and collect the supernatant and precipitate separately for later use;

[0073] (4) Repeat steps (2)-(3) and combine the supernatants collected twice.

[0074] (5) The solvent was concentrated by nitrogen blowing, and the sample of Anoectochilus roxburghii-derived nanovesicles was obtained by filtration and stored for later use.

[0075] The ultrasound parameters in step (2) are: 500W, 15min.

[0076] The storage temperature in step (4) is 4℃.

[0077] The mobile phase was methanol-0.3% phosphoric acid aqueous solution (v / v 55:45); the column temperature was 30℃; quercetin standard (A) and the ultrasonic extract of *Anoectochilus roxburghii* nanovesicles (B) were analyzed at 375 nm. The chromatograms are shown below. Figure 2 As shown.

[0078] Example 3

[0079] The cytotoxicity of quercetin standard and Anoectochilus roxburghii-derived nanovesicles was verified using the MTT assay. The specific procedure is as follows:

[0080] (1) The cell suspension of human liver cancer cells HepG2 was evenly seeded into a 96-well plate, and the culture medium was aspirated after the cells adhered to the plate.

[0081] (2) Administration: Quercetin standard and Anoectochilus roxburghii-derived nanovesicles from Example 1 were added to different wells of the above 96-well plate, and incubated in a 37°C incubator for 24 hours before being aspirated.

[0082] (3) Add MTT solution to the above 96-well plate, react for 4 hours and then remove it;

[0083] (4) Add dimethyl sulfoxide (DMSO) to the above 96-well plate, shake, and measure the absorbance with an enzyme-linked immunosorbent assay (ELISA) reader to calculate the cell viability.

[0084] In step (1), the cell suspension concentration is 5000 cells / well, and the cell suspension volume per well is 200 μL;

[0085] In step (2), the dosage concentrations of quercetin standard are 0.35 ng / mL and 0.70 ng / mL; the dosage concentrations of Anoectochilus roxburghii-derived nanovesicles are 25 ug / mL and 50 ug / mL (containing quercetin concentrations of 0.35 ng / mL and 0.70 ng / mL, respectively).

[0086] In step (3), the concentration of MTT is 5 mg / mL, and the volume added to each well is 20 μL.

[0087] In step (4), the volume of DMSO is 150 μL and the detection wavelength is 570 nm.

[0088] like Figure 3 As shown, after co-incubating HepG2 cells with *Anoectochilus roxburghii*-derived nanovesicles at concentrations of 25 μg / mL and 50 μg / mL (containing quercetin concentrations of 0.35 ng / mL and 0.70 ng / mL, respectively) and the corresponding concentrations of quercetin for 24 hours, the cell survival rate did not change significantly (P>0.05). This indicates that the experimental doses of *Anoectochilus roxburghii*-derived nanovesicles and quercetin had no significant toxic side effects on HepG2 cells, making them suitable for use as therapeutic agents in the treatment of alcoholic cell damage.

[0089] Example 4

[0090] The MTT assay was used to verify the effect of quercetin standard and Anoectochilus roxburghii-derived nanovesicles on the resistance of HepG2 cells to alcohol damage. The specific procedure is as follows:

[0091] (1) HepG2 cell suspension was inoculated into 96-well plates. After the cells adhered, the culture medium was aspirated. Drug administration group, alcohol group and blank control group were set up.

[0092] (2) Administration: Quercetin standard and Anoectochilus roxburghii-derived nanovesicles from Example 1 were added to the administration groups, and incubated in a 37°C incubator for 24 hours before being aspirated; except for the blank control group, 5% alcohol was added to the other groups, and PBS was added to the blank control group, and incubated in a 37°C incubator for 24 hours before being aspirated.

[0093] (3) Add MTT solution to the above 96-well plate, react for 4 hours and then remove it;

[0094] (4) Add dimethyl sulfoxide (DMSO) to the above 96-well plate, shake, and measure the absorbance with an enzyme-linked immunosorbent assay (ELISA) reader to calculate the cell viability.

[0095] In step (1), the cell suspension concentration is 5000 cells / well, and the cell suspension volume per well is 200 μL;

[0096] In step (2), the dosage concentrations of quercetin standard are 0.35 ng / mL and 0.70 ng / mL; the dosage concentrations of Anoectochilus roxburghii-derived nanovesicles are 25 ug / mL and 50 ug / mL (containing quercetin concentrations of 0.35 ng / mL and 0.70 ng / mL, respectively).

[0097] In step (3), the concentration of MTT is 5 mg / mL, and the volume added to each well is 20 μL.

[0098] In step (4), the volume of DMSO is 150 μL and the detection wavelength is 570 nm.

[0099] like Figure 4 As shown, compared with the blank control group, the cell survival rate in the alcohol group was significantly decreased. Compared with the alcohol group, the cell survival rate of both the *Anoectochilus roxburghii*-derived nanovesicle group and the quercetin standard group was improved to varying degrees, and the *Anoectochilus roxburghii*-derived nanovesicle group showed a more significant effect on improving cell survival rate.

[0100] Example 5

[0101] Twenty-one healthy male ICR mice weighing 20-24g were randomly divided into a blank control group, an alcohol group, and an administration group. The administration groups included ADNVs at doses of 50mg / kg and 25mg / kg (containing quercetin concentrations of 0.70μg / kg and 0.35μg / kg, respectively), corresponding doses of quercetin standards (0.70μg / kg and 0.35μg / kg), and a silymarin (50mg / kg) positive control group, with three mice in each group. After three days of acclimatization, the mice were administered the drugs via gavage once daily. The administration groups received high and low doses of ADNVs, corresponding concentrations of quercetin standards, and silymarin, respectively. The control and alcohol groups received an equal volume of PBS daily. An acute alcoholic liver injury model was established after 9 days. Two hours after administration on the ninth day, all mice except the blank control group received 50% ethanol (12mL / kg) via gavage. After ethanol gavage, mice were kept from eating but allowed to drink water. They were weighed 12 hours later, and their livers were collected for pathological observation.

[0102] Mice were euthanized immediately after blood collection, and the livers were separated. After rinsing in physiological saline, the surface of the liver tissue was blotted dry with filter paper. Liver lesions were visually observed, and the liver weight was weighed and recorded. Finally, liver tissue sections were prepared and stained with hematoxylin-eosin (H&E) for observation. The steps for preparing pathological sections are as follows:

[0103] S1: Immerse the tissue in 4% paraformaldehyde for 24 hours for fixation. Remove the fixed material, trim it to make the cut surface smooth, and dehydrate and clear it. Then place the cleared tissue block in melted paraffin and keep it in a paraffin bath for insulation. After the paraffin has completely immersed the tissue block, embed and section it. Float the sections on the surface of 40℃ warm water to flatten them, then use a slide with anti-detachment treatment to retrieve them, bake the slides, and store them.

[0104] S2: The paraffin in the sections was removed using xylene, then stained with hematoxylin-eosin solution, and then separated and dehydrated for clearing.

[0105] In step S1, the dehydration and clearing treatment sequence is as follows: dehydration overnight in 75% ethanol, dehydration for 2 hours in 85% ethanol, dehydration for 2 hours in 90% ethanol, dehydration for 1 hour in 95% ethanol, dehydration for 30 minutes in 100% ethanol I, and dehydration for 30 minutes in 100% ethanol II; then the tissue is cleared in 1 / 2 anhydrous ethanol + 1 / 2 xylene for 5-10 minutes, cleared in xylene I for 5-10 minutes, and cleared in xylene II for 5-10 minutes; the section thickness is 4 μm;

[0106] In step S2, the specific process of dewaxing xylene is as follows: dewaxing in xylene I for 10 min; dewaxing in xylene II for 10 min; b. dewaxing in 100% ethanol I for 5 min; dewaxing in 100% ethanol II for 5 min; ethanol in 95% ethanol for 5 min; ethanol in 85% ethanol for 5 min; ethanol in 75% ethanol for 5 min; and then washing away the ethanol with distilled water for 5 min.

[0107] like Figure 5 As shown, in the blank control group, hepatocytes had clear boundaries and visible sinusoids; in the alcohol group, hepatocytes were edematous, sinusoids were compressed and narrowed, cell boundaries were indistinct, and fat vacuoles were enlarged and increased in number, indicating hepatocyte steatosis and significantly increased lipid accumulation in the liver. Compared with the alcohol group, the *Anoectochilus roxburghii*-derived nanovesicles and silymarin treatment groups showed varying degrees of improvement in hepatocyte edema and steatosis, with clear cell boundaries and a moderate increase in lipid accumulation. However, the two doses of quercetin standard treatment groups showed no significant improvement in hepatocyte steatosis, and lipid accumulation remained unchanged compared to the alcohol group. These results indicate that *Anoectochilus roxburghii*-derived nanovesicles have a preventive and protective effect against acute alcohol-induced liver tissue damage, and can also prevent the formation of fatty liver under alcohol stimulation.

Claims

1. The application of *Anoectochilus roxburghii*-derived nanovesicles in the preparation of drugs for the prevention and treatment of alcoholic liver disease, characterized in that: The preparation method of the *Anoectochilus roxburghii*-derived nanovesicles includes the following steps: S1: Juice the fresh whole plant of Anoectochilus roxburghii using a juicer, filter it with gauze, and obtain the filtrate; S2: Centrifuge the filtrate obtained in step S1 at high speed, discard the precipitate, and obtain the supernatant; S3: The supernatant obtained in step S2 was centrifuged at high speed, the precipitate was collected and resuspended in PBS to obtain a suspension of Anoectochilus roxburghii-derived nanovesicles containing quercetin and which can protect against alcoholic liver injury. The juicing process requires mixing the fresh whole plant of *Anoectochilus roxburghii* with pre-cooled PBS or PBS ice cubes. The alcoholic liver disease mentioned above refers to alcoholic liver injury and alcoholic fatty liver.

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