Preparation method and application of ginseng-sourced nano-like particle loaded medicine

Abstract

The invention relates to the technical field of biological medicines and nano preparations, in particular to a preparation method and application of a ginseng-sourced nano-like particle loaded medicine. The method comprises the following steps: S1, extracting a ginseng exosome; s2, purifying the ginseng exosome; and S3, preparing the drug-loaded nanoparticles. According to the invention, GENs is taken as an oral carrier, the problem of low bioavailability of free drugs is solved, the drug stability is enhanced by a natural phospholipid double-layer structure, and intestinal absorption and liver targeting accumulation are facilitated. The dual effects of the active ingredient of the GENs and the loaded drug are obviously better than that of a single drug; and oxidation resistance, inflammation resistance, fibrosis resistance and apoptosis resistance are synchronously realized. By repairing ZO-1 / Occludin, an intestinal barrier is reconstructed, intestinal toxin entering the liver is reduced, and meanwhile, liver injury and fibrosis disappearance are improved; a new strategy is provided for high-valued transformation of ginseng resources, and the safety of the plant exosome is far higher than that of a synthetic vector.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and nanoparticle technology, specifically to a method for preparing and applying a drug loaded with ginseng-derived nanoparticles. Background Technology

[0002] Liver fibrosis is a key pathological process in the progression of chronic liver disease to cirrhosis and hepatocellular carcinoma. Liver-related mortality increases exponentially with increasing fibrosis. It is characterized by increased extracellular matrix (ECM) synthesis due to hepatic stellate cell activation, leading to excessive ECM deposition. In advanced stages, it can develop into cirrhosis and even hepatocellular carcinoma, with end-stage patients experiencing liver failure and death. Early-stage liver fibrosis often presents with few or no clinical symptoms, and diagnosis is usually made in the middle or late stages. It is characterized by its high severity and difficulty in reversing disease. Its incidence is rising globally, particularly among high-risk groups such as those with chronic viral hepatitis (e.g., hepatitis B and C), non-alcoholic fatty liver disease (MAFLD), and alcoholic liver disease.

[0003] Liver fibrosis is mainly caused by the activation of hepatic stellate cells, leading to increased extracellular matrix deposition and hardening of the liver, which can further develop into cirrhosis and even liver cancer. The pathological process of liver fibrosis is complex, involving multiple liver cells, and simply inhibiting the activation of hepatic stellate cells is a limited approach; moreover, the utilization rate of orally administered free drugs is low and there is attrition, while the efficacy of single drugs is limited. Summary of the Invention

[0004] The purpose of this invention is to provide a ginseng-derived nanoparticle-loaded drug, its preparation method, and its application. By loading the drug onto ginseng exosomes, the circulation of the gut-hepatic axis is utilized to coordinate the regulation of the intestinal flora, thereby alleviating liver fibrosis and solving the problems mentioned in the background art.

[0005] To achieve the above objectives, on the one hand, the present invention provides a method for preparing a drug loaded with ginseng-derived nanoparticles, comprising the following steps: S1. Extraction of ginseng exosomes (GENs): Wash the ginseng, cut it into small pieces, add PBS buffer, homogenize it using a homogenizer, filter it through gauze, and centrifuge it at 3000g for 30 min and 10000g for 1 h in sequence. Take the supernatant and centrifuge it at 100000g for 1 h in an ultracentrifuge. Take the precipitate, resuspend the precipitate in PBS, and vortex it thoroughly to obtain crude ginseng exosomes (GENs). S2. Purification of Ginseng Exosomes (GENs): Prepare 1 mol / L and 2 mol / L sucrose solutions. Place the resuspended sample in an ultracentrifuge tube, and then sequentially pass the sample layer through the 1 mol / L and 2 mol / L sucrose solutions. Centrifuge at 150,000g for 2 h, aspirate the sample layer, add PBS to the entire tube, centrifuge at 100,000g for 1 h, wash away excess sucrose, and resuspend the precipitate with PBS to obtain purified ginseng exosomes. The protein concentration in the ginseng exosomes was determined using a BCA kit and stored at -80℃. S3. Preparation of drug-loaded exosomes: Ginseng exosome (GENs) solution was mixed with free drug (preferably ferulic acid FA) at a mass ratio of 1:2-4, and the mixture was sonicated intermittently for 1 hour, followed by overnight incubation on a shaker at 4°C to obtain ginseng-derived nanoparticle-loaded drug (Exo@FA) (the nanoparticle-loaded drug has a particle size of 150-200 nm and a surface potential of -25 to -30 mV).

[0006] This invention utilizes ginseng exosomes (GENs) as a natural carrier. After purification via sucrose density gradient centrifugation, drugs (such as ferulic acid FA) are loaded onto the GENs, forming Exo@FA nanoparticles with a particle size of approximately 191 nm. After oral administration, the GENs enhance drug bioavailability and target delivery to the liver. Containing active ingredients such as ginsenosides, the GENs synergistically exert anti-inflammatory and antioxidant effects with the loaded drug. Liver protection: Reduces intracellular ROS levels in hepatocytes (H2O2 model: fluorescence intensity decreased from 92.1% to 23.7%; LPS model: decreased from 16.7% to 4.90%), inhibits apoptosis (apoptosis rate decreased from 73.7% to ≤5%) and enhances cell viability (CCK-8 proliferation rate significantly increased). Anti-fibrosis: Inhibits the expression of Vimentin / α-SMA, a marker of hepatic stellate cell fibrosis (disappearance of green fluorescence), and reduces collagen deposition (Sirius red / Masson staining reversal).

[0007] Secondly, GENs repair the intestinal barrier by regulating the gut microbiota: restoring the integrity of intestinal villi structure and intestinal wall thickness; upregulating the expression of tight junction proteins ZO-1 and Occludin, thus improving intestinal permeability. The repaired intestinal barrier reduces endotoxin entry into the bloodstream, indirectly improving the inflammatory microenvironment of the liver, forming a positive cycle of "intestinal repair → liver protection".

[0008] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Preparation method and application of ginseng-derived nanoparticle-loaded drugs: GENs serve as oral carriers to address the problem of low bioavailability of free drugs. Their natural phospholipid bilayer structure enhances drug stability, and their particle size (191 nm) and potential (-29.0 mV) facilitate intestinal absorption and liver-targeted accumulation.

[0009] 2. In the preparation method and application of the drug loaded on ginseng-derived nanoparticles, the dual effect of the active ingredients of GENs and the loaded drug (FA) is significantly stronger than that of single drug (Exo@FA / FA: ROS reduction of +40.7%); simultaneously achieving anti-oxidation (ROS reduction), anti-inflammation (TNF-α / IL-6 reduction), anti-fibrosis (α-SMA reduction) and anti-apoptosis (92.7% increase in live cell ratio, flow cytometry data).

[0010] 3. The preparation method and application of the drug loaded on the ginseng-derived nanoparticles demonstrate that by repairing ZO-1 / Occludin to rebuild the intestinal barrier, the entry of enterogenic toxins into the liver is reduced, while liver damage (AST / ALT reduction >50%) and fibrosis (hepatic nodules) disappearance are improved; this provides a new strategy for the high-value transformation of ginseng resources, and the safety of plant exosomes is much higher than that of synthetic carriers. Attached Figure Description

[0011] Figure 1 This is a schematic diagram of the particle size of GENs before and after drug loading in Example 1 of the present invention; Figure 2 This is a schematic diagram of the potentials of GENs before and after drug loading in Embodiment 1 of the present invention; Figure 3 Transmission electron microscope (TEM) image of the GENs in Embodiment 1 of the present invention; Figure 4 This is the ROS fluorescence image of Experimental Example 1 of the present invention; Figure 5 This is the ROS fluorescence quantification chromatogram of Experimental Example 1 of the present invention; Figure 6 This is a bar chart of CCK-8 screening H2O2 dosage for Experiment Example 2 of the present invention; Figure 7 This is a bar chart comparing the CCK-8 screening results of Experiment Example 2 of this invention. Figure 8 This is the staining fluorescence image of Experimental Example 2 of the present invention; Figure 9 This is a bar graph showing the TNF-α secretion and IL-1β secretion of hepatocytes in Experiment Example 3 of the present invention; Figure 10 The bar chart shows the TNF-α secretion and IL-1β secretion of RAW in Experimental Example 3 of the present invention. Figure 11 The fluorescence spectrum of Vimentin content in Experimental Example 4 of this invention is shown. Figure 12 This is a quantitative graph of Vimentin content in Experimental Example 4 of the present invention; Figure 13 The fluorescence spectrum of α-SMA content in Experimental Example 4 of this invention is shown. Figure 14 This is a quantitative chromatogram of α-SMA content in Experimental Example 4 of the present invention; Figure 15 This is the ROS fluorescence image of Experimental Example 5 of the present invention; Figure 16 This is the fluorescence quantitative chromatogram of Experimental Example 5 of the present invention; Figure 17 This is a schematic diagram of the gross liver of each group of mice in Experiment Example 6 of the present invention; Figure 18 The fluorescence diagram shows the content of two fibrinogenic proteins in Experimental Example 6 of this invention. Figure 19 The images show HE, Sirius red, and Masson stained sections of mouse liver from Experiment Example 6 of this invention. Figure 20 This is a bar chart of mouse serum AST levels from Experiment Example 6 of the present invention; Figure 21 This is a bar graph of mouse serum ALT levels from Experiment Example 6 of the present invention; Figure 22 HE slices of mouse duodenum and jejunum from Experiment Example 7 of this invention; Figure 23 The following are statistical diagrams of duodenal and jejunal intestinal wall thickness in Experiment Example 7 of this invention; Figure 24 This is a graph showing the content of ZO-1 protein in the duodenum in Experiment Example 7 of the present invention; Figure 25 The content of duodenal ocludin protein in Experimental Example 7 of the present invention; Figure 26 This is a graph showing the content of ZO-1 protein in the jejunum in Experiment Example 7 of the present invention; Figure 27 This is a graph showing the content of jejunal Occludin protein in Experimental Example 7 of the present invention. Detailed Implementation

[0012] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0013] Example 1: This embodiment of the invention provides a method for preparing a drug loaded with ginseng-derived nanoparticles, comprising the following steps: S1. Ginseng exosome extraction: Wash the ginseng, cut it into small pieces, add an appropriate amount of PBS buffer, homogenize it using a homogenizer, filter it through gauze, and centrifuge it at 3000g for 30 min and 10000g for 1 h in sequence. Take the supernatant and centrifuge it at 100000g for 1 h using an ultracentrifuge. Take the precipitate, resuspend the precipitate in PBS, and vortex it thoroughly to obtain the crudely extracted ginseng exosomes. S2. Purification of ginseng exosomes: Prepare 1 mol / L and 2 mol / L sucrose solutions. Place the resuspended sample in an ultracentrifuge tube, and then sequentially pass the sample layer through the 1 mol / L and 2 mol / L sucrose solutions. Centrifuge at 150,000g for 2 h, aspirate the sample layer, add PBS to the entire tube, centrifuge at 100,000g for 1 h, wash away excess sucrose, and resuspend the precipitate with PBS to obtain purified ginseng exosomes. The protein concentration in the ginseng exosomes was determined using a BCA kit and stored at -80℃. S3. Preparation of drug-loaded exosomes: The ginseng exosome solution and free drug were mixed at a ratio of 1:3, intermittently sonicated for 1 hour, and then incubated overnight on a shaker at 4°C.

[0014] according to Figure 1 As shown, the particle size of GENs before and after drug loading is ±162.3 nm before drug loading and ±191 nm after drug loading. According to... Figure 2 As shown, the potentials of GENs before and after drug loading are: average potential before drug loading -35.5, average potential after drug loading -29.0. According to... Figure 3 As shown in the transmission electron microscope image of GENs, a clear bilayer film structure is evident.

[0015] Experimental Example 1: In vitro hepatocyte antioxidant test (1) ROS release from hepatocytes: The cells were divided into five groups, namely: Control group; H2O2 group; GENS group: H2O2 + GENs; FA group: H2O2+FA; Exo@FA group: H2O2+Exo@FA.

[0016] Test method: 24 hours after each group was administered the drug, the culture medium was discarded, the cells were rinsed with PBS, DHE dye was added, the dye was washed off, Hoechst nuclear dye was added, the cells were rinsed with PBS, and the cells were observed and photographed under a fluorescence microscope.

[0017] The ROS fluorescent probe, dihydroethidium (DHE), can freely penetrate the cell membrane and enter the cell, where it is oxidized by ROS to form ethidium oxide. Ethidium oxide can be incorporated into chromosomal DNA, producing red fluorescence. The amount and changes in cellular ROS content can be determined based on the production of red fluorescence in living cells.

[0018] according to Figure 4 and Figure 5 As shown, compared with the Control group, the red fluorescence intensity of the H2O2 group was significantly increased, indicating that the generation of reactive oxygen species increased. The fluorescence intensity of the GENs group and FA group was weaker than that of the H2O2 group, indicating that the generation of ROS decreased after administration. In the Exo@FA group, the red fluorescence was almost invisible after administration, indicating that its antioxidant effect was better than that of administration alone.

[0019] (2) ROS release from hepatocytes The cells were divided into five groups, namely: Control group; LPS group; GENs group: LPS + GENs; FA group: LPS+FA; Exo@FA group: LPS + Exo@FA.

[0020] Test method: 24 hours after each group was administered the drug, the culture medium was discarded, the cells were rinsed with PBS, DHE dye was added, the dye was eluted, and the cells were resuspended in PBS. The release of ROS was detected by flow cytometry using an appropriate fluorescence channel.

[0021] According to experimental data, the fluorescence intensity of the Control group was only 0.39%. Compared with the Control group, the fluorescence intensity of the LPS group increased significantly to 92.1%, indicating increased reactive oxygen species (ROS) generation. The fluorescence intensity of the GENs group and the FA group was weaker than that of the LPS group, decreasing to 68.8% and 64.4% respectively, indicating reduced ROS generation after administration. The fluorescence intensity of the Exo@FA group was 23.7% after administration, indicating that its antioxidant effect was better than that of administration alone.

[0022] Experimental Example 2: In vitro hepatocyte damage repair test (1) Effects of different preparations on hepatocyte repair: The cells were divided into five groups, namely: Control group; H2O2 group; GENs group: H2O2 + GENs; FA group: H2O2+FA; Exo@FA group: H2O2+Exo@FA.

[0023] Test method: After 24 hours of drug administration to each group, the culture medium was discarded, a certain proportion of CCK-8 reagent was added, and cell viability was detected by microplate reader after a certain period of time.

[0024] according to Figure 6 and Figure 7 As shown, compared with the Control group, the cell proliferation activity in the H2O2 group was significantly reduced; while the activity of the GENs group and FA group increased after administration alone compared with the H2O2 group, indicating that cell damage was alleviated and repaired. The cell proliferation activity in the Exo@FA group was the most significant, indicating that it can further enhance tissue cell repair and has a more obvious effect.

[0025] (2) Effects of different formulations on hepatocyte apoptosis: The cells were divided into five groups, namely: Control group; H2O2 group; GENs group: H2O2 + GENs; FA group: H2O2+FA; Exo@FA group: H2O2+Exo@FA.

[0026] Test method: Cells were collected 24 hours after each group was administered the drug, and the cell pellet was added with apoptosis reagent. The apoptosis rate was detected by flow cytometry.

[0027] According to the experimental data, the viable cell ratio in the Control group was 92.72%, with an early apoptosis rate of 3.91% and a late apoptosis rate of 2.36%. After the addition of H2O2, the viable cell ratio decreased to 10.12%, with early apoptosis at 48.79% and late apoptosis at 24.91%, indicating that the cells were damaged and died. After GENs and FA were administered alone, the viable cell ratio increased and the apoptosis rate decreased. However, the Exo@FA administration group had the highest viable cell ratio and the lowest apoptosis rate, essentially returning to normal levels.

[0028] (3) Effects of different formulations on hepatocyte viability The cells were divided into five groups, namely: Control group; H2O2 group; GENs group: H2O2 + GENs; FA group: H2O2+FA; Exo@FA group: H2O2+Exo@FA.

[0029] Test method: 24 hours after each group was administered the drug, the culture medium was discarded, PBS was added for rinsing, AM / PI live / dead staining solution was added for incubation, and finally PBS was rinsed. The ratio of live to dead cells was observed under a fluorescence microscope.

[0030] Staining principle: Calcein-AM is a cell-permeable dye that is itself non-fluorescent. Upon entering a living cell, Calcein-AM is hydrolyzed by intracellular esterases, producing Calcein, which has strong green fluorescence. PI is a nucleic acid dye that cannot penetrate intact cell membranes, but can enter cells with damaged or dead membranes. PI binds to DNA in the cell nucleus and emits red fluorescence.

[0031] according to Figure 8 As shown, under the same fluorescence conditions, the Control group showed almost no red fluorescence, and all cells in the field of view were live. The H202 group showed obvious red fluorescent spots, indicating an increase in the number of dead cells. After administration to the GENs and FA groups, the number of fluorescent spots of dead cells in the field of view decreased. However, after administration to the Exo@FA group, the red fluorescent spots were almost invisible, indicating that the proportion of dead cells was the lowest and the repair ability after damage was the strongest.

[0032] Experimental Example 3: In vitro hepatocyte anti-inflammatory test (1) Effects of different formulations on inflammatory factors secreted by cells: The cells were divided into five groups, namely: Control group; LPS group; GENs group: LPS + GENs; FA group: LPS+FA; Exo@FA group: LPS + Exo@FA.

[0033] Test method: Cell culture medium was collected 24 hours after drug administration in each group, and the supernatant was obtained by centrifugation. The content of inflammatory factors in the supernatant of each group was detected by ELISA kit.

[0034] according to Figure 9 As shown, hepatocytes release inflammatory factors after stimulation with a certain concentration of LPS. The Exo@FA group showed the lowest secretion of inflammatory factors after administration, exhibiting the best anti-inflammatory effect and effectively reducing the secretion of inflammatory factors. According to... Figure 10 As shown, RAW releases inflammatory factors after stimulation with a certain concentration of LPS. The Exo@FA group secreted the fewest inflammatory factors after administration, exhibiting the best anti-inflammatory effect and effectively reducing the secretion of inflammatory factors.

[0035] Experimental Example 4: In vitro anti-fibrotic test (1) Immunofluorescence: Effects of different formulations on fibrin secretion by hepatic stellate cells: The cells were divided into five groups, namely: Control group; TGF-β group; GENs group: TGF-β+GENs; FA group: TGF-β+FA; Exo@FA group: TGF-β+Exo@FA.

[0036] Test method: 24 h after each group was administered the drug, the culture medium was discarded, PBS was added for rinsing, and the cells were fixed with 4% paraformaldehyde for 15 min, followed by PBS washing 2-3 times; the membrane was perforated with 0.1% Triton for 15 min, followed by PBS washing 2-3 times; after blocking with blocking solution, the cells were washed; primary antibody was added and incubated at 4°C overnight; after washing, fluorescent secondary antibody was added and incubated in the dark for one hour; after washing with PBS, nuclear dye was added, and after staining, the cells were washed with PBS and observed and photographed using a fluorescence microscope.

[0037] During fibrosis, both Vimentin and α-SMA are closely related to cell activation and functional changes. For example, in liver fibrosis, after hepatic stellate cells are activated, Vimentin expression increases, and the cell morphology changes from a quiescent state to an activated state; at the same time, α-SMA expression also increases significantly, and the cells acquire contractile function, promoting the development of liver fibrosis.

[0038] according to Figure 11 and Figure 12 As shown, TGF-β stimulation increased Vimentin expression, while administration of drugs to the GENs and FA groups decreased Vimentin expression. In the Exo@FA group, green fluorescence was almost invisible after drug administration. According to... Figure 13 and Figure 14 As shown, TGF-β stimulation significantly increased α-SMA expression, while GENs and FA groups showed decreased α-SMA expression. In the Exo@FA group, green fluorescence was almost absent after administration, indicating that it had the strongest anti-fibrotic effect.

[0039] Experimental Example 5: In vitro hepatic stellate cell antioxidant assay (1) ROS release from hepatic stellate cells: The cells were divided into five groups, namely: Control group; H2O2 group; GENs group: H2O2 + GENs; FA group: H2O2+FA; Exo@FA group: H2O2+Exo@FA.

[0040] Test method: 24 hours after each group was administered the drug, cells were treated with a suitable fluorescent dye and then observed and photographed under a fluorescence microscope.

[0041] The ROS fluorescent probe, dihydroethidium (DHE), can freely penetrate the cell membrane and enter the cell, where it is oxidized by ROS to form ethidium oxide. Ethidium oxide can be incorporated into chromosomal DNA, producing red fluorescence. The amount and changes in cellular ROS content can be determined based on the production of red fluorescence in living cells.

[0042] according to Figure 15 As shown, compared with the Control group, the red fluorescence intensity of the H2O2 group was significantly increased, indicating that the generation of reactive oxygen species increased. The fluorescence intensity of the GENs group and the FA group was weaker than that of the H2O2 group, indicating that the generation of ROS was reduced after administration. The red fluorescence of the Exo@FA group was almost invisible after administration, indicating that its antioxidant effect was better than that of administration alone.

[0043] (2) ROS release from hepatic stellate cells: The cells were divided into five groups, namely: Control group; LPS group; GENs group: LPS + GENs; FA group: LPS+FA; Exo@FA group: LPS + Exo@FA.

[0044] Test method: 24 hours after each group was administered the drug, the culture medium was discarded, the cells were rinsed with PBS, DHE dye was added, the dye was eluted, and the cells were resuspended in PBS. The release of ROS was detected by flow cytometry using an appropriate fluorescence channel.

[0045] according to Figure 16 As shown, the fluorescence intensity of the Control group was only 1.36%. Compared with the Control group, the fluorescence intensity of the H2O2 group increased significantly to 16.7%, indicating that the generation of reactive oxygen species increased. The fluorescence intensity of the GENs group and FA group was weaker than that of the LPS group, decreasing to 11.1% and 7.66% respectively, indicating that the generation of ROS decreased after administration. The fluorescence intensity of the Exo@FA group was 4.90% after administration, indicating that its antioxidant effect was better than that of administration alone.

[0046] Experimental Example 6: In vivo fibrosis test (1) Divide the mice into 5 groups: Healthy group: fed normally, without any treatment; CCl4 group: 20% CCl4 was injected intraperitoneally three times a week; GENs group: intraperitoneal injection of CCl4 followed by gavage administration of GENs (20 mg / ml); FA group: intraperitoneal injection of CCl4 followed by gavage administration of FA (20 mg / ml); Exo@FA group: CCl4 was injected intraperitoneally and Exo@FA was administered by gavage.

[0047] Test method: Four weeks after modeling, each group was given gavage treatment three times a week. Organs were collected from mice after four weeks.

[0048] according to Figure 17 As shown, the livers in the Control group were rosy and glossy with a smooth surface, while the livers in the Model group had irregular nodules and a granular texture. After administration of the Exo@FA group, the number of nodules decreased and the granular texture on the surface disappeared, indicating that it had the best therapeutic effect.

[0049] according to Figure 18 As shown, the Control group showed almost no green fluorescence; the model group showed a large amount of protein accumulation around the liver lobules and central vein, with significantly enhanced green fluorescence; and the Exo@FA group showed a significant reduction in protein deposition after administration.

[0050] according to Figure 19 As shown: HE: The control group showed normal liver structure, clear lobular structure, neatly arranged hepatocytes, uniform cytoplasm, clearly visible nuclei, and no obvious abnormalities around the central vein; the model group showed disordered hepatocyte arrangement and obvious inflammatory cell infiltration; the Exo@FA group showed that hepatocytes returned to normal levels after treatment.

[0051] Sirius red staining: In the Control group, the liver structure was normal, and collagen fibers were evenly distributed, mainly concentrated around the central vein and the edge of the liver lobules; in the Model group, a large amount of collagen fibers were deposited, forming bridging fibrosis, the liver lobule structure was destroyed, and the fibrous septa were widened; in the Exo@FA group, hepatocytes recovered to normal levels after treatment.

[0052] Masson staining: In the Control group, the distribution of collagen fibers in the liver was normal, mainly concentrated around the central vein and the edge of the liver lobule, and stained blue; in the Model group, a large amount of collagen fibers were deposited, forming obvious fibrous septa, and the structure of the liver lobule was destroyed, and the fibrous septa were blue; in the Exo@FA group, hepatocytes recovered to normal levels after treatment.

[0053] (2) Divide the mice into 5 groups: Healthy group: fed normally, without any treatment; CCl4 group: 20% CCl4 was injected intraperitoneally three times a week; GENs group: intraperitoneal injection of CCl4 followed by gavage administration of GENs (20 mg / ml); FA group: intraperitoneal injection of CCl4 followed by gavage administration of FA (20 mg / ml); Exo@FA group: CCl4 was injected intraperitoneally and Exo@FA was administered by gavage.

[0054] Test method: Four weeks after modeling, each group was given gavage treatment three times a week. Serum was collected from mice after four weeks.

[0055] according to Figure 20 and Figure 21 As shown, compared with the Control group, the serum AST and ALT levels of mice in the model group were significantly elevated, indicating hepatocellular damage and liver lesions. After treatment with Exo@FA, the AST and ALT levels in the Exo@FA group were significantly reduced, indicating that hepatocellular damage was alleviated and liver function was restored. AST and ALT are two commonly used indicators of liver function, mainly used to assess liver health. They are released from hepatocellular cells into the bloodstream when the liver is damaged; therefore, elevated serum AST and ALT levels usually indicate hepatocellular damage.

[0056] Experimental Case 7: Repair of Intestinal Barrier Damage (1) Divide the mice into 5 groups: Healthy group: fed normally, without any treatment; CCl4 group: 20% CCl4 was injected intraperitoneally three times a week; GENs group: intraperitoneal injection of CCl4 followed by gavage administration of GENs (20 mg / ml); FA group: intraperitoneal injection of CCl4 followed by gavage administration of FA (20 mg / ml); Exo@FA group: intraperitoneal injection of CCl4 followed by gavage administration of Exo@FA; Test method: Four weeks after modeling, each group was given gavage treatment three times a week. After four weeks, the duodenal and jejunal tissues of the mice were collected.

[0057] according to Figure 22 As shown, compared with the Control group, the arrangement and integrity of intestinal villi in the model group mice were disrupted, which was improved after administration of Exo@FA.

[0058] according to Figure 23 As shown, the intestinal wall of mice was damaged and its thickness was reduced after modeling, and the intestinal wall thickness returned to normal after treatment.

[0059] according to Figure 24-27 As shown, in the intestines of healthy mice, ZO-1 and Occludin form a continuous distribution at the tight junctions of intestinal epithelial cells, maintaining the integrity of the intestinal barrier. However, in model mice, the expression levels of ZO-1 and Occludin may be significantly downregulated, and their distribution becomes discontinuous, leading to impaired tight junction integrity and increased intestinal permeability; the damage is alleviated and repaired after treatment. Occludin works synergistically with ZO-1 in the tight junctions of intestinal epithelial cells to maintain intestinal barrier function. Under healthy conditions, Occludin forms a continuous distribution at the tight junctions between cells.

[0060] Furthermore, at the door level: Bacteroidota: Increased in proportion in the model group, decreased to healthy levels after treatment. Bacteroid bacteria (such as the genus Bacteroides) are Gram-negative bacteria whose cell walls contain lipopolysaccharide (LPS).

[0061] Firmicutes are the dominant phylum of bacteria in the gut, with a high proportion in the healthy group, decreasing in the model group, and returning to healthy levels after treatment. Their dominant genera, such as Lactobacillus, produce lactic acid and antimicrobial peptides, inhibiting enterogenic pathogens and reducing LPS translocation. An increased B / F ratio, excessive proliferation of LPS-producing Bacteroides, and a decrease in butyrate-producing bacteria weaken the intestinal barrier protection.

[0062] Cyanobacteria: Their proportion increased in the model group and decreased after treatment. Genera associated with liver fibrosis: 1. *Microcystis*: Secretes microcystin (MCs), which directly damages hepatocytes, induces oxidative stress and inflammatory responses, and promotes hepatic stellate cell (HSC) activation, associated with cirrhosis and liver cancer. 2. *Anabaena*: Some strains produce toxins that exacerbate intrahepatic inflammation by activating the TLR4 / NF-κB pathway, indirectly promoting fibrosis. Mechanism of action: Toxins induce hepatocyte apoptosis, release damage-associated molecular patterns (DAMPs), activate HSCs and macrophages, and induce collagen deposition.

[0063] p_Actinobacteriota (Actinomycetes): The proportion decreased in the model group but increased after treatment. Related genus: Bifidobacterium. Protective effects: Secretes acetic acid and lactic acid, lowers intestinal pH, inhibits the growth of pathogenic bacteria, and reduces endotoxin production. Enhances the integrity of the intestinal mucus layer, reduces LPS translocation, and alleviates intrahepatic inflammation.

[0064] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely preferred examples and are not intended to limit the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a drug loaded with ginseng-derived nanoparticles, characterized in that, Includes the following steps: S1. Ginseng exosome extraction: Wash the ginseng, cut it into small pieces, add PBS buffer, homogenize it using a homogenizer, filter it with gauze, centrifuge to collect the supernatant, centrifuge again using an ultracentrifuge, collect the precipitate, resuspend the precipitate with PBS, and vortex thoroughly to obtain crude extracted ginseng exosomes. S2. Purification of ginseng exosomes: The crude ginseng exosomes were placed in a sucrose density gradient solution, centrifuged, and the sample layer was aspirated. After adding PBS buffer, the sample was centrifuged and washed. The precipitate was resuspended in PBS to obtain pure ginseng exosomes. S3. Preparation of drug-loaded nanoparticles: The purified ginseng exosome solution and free drug were mixed in a mass ratio, and the mixture was subjected to intermittent sonication and incubated overnight in a shaker to obtain drug-loaded nanoparticles.

2. The method for preparing ginseng-derived nanoparticle-loaded drugs according to claim 1, characterized in that: The free drug is ferulic acid.

3. The method for preparing ginseng-derived nanoparticle-loaded drugs according to claim 1, characterized in that: The ginseng exosome solution and free drug are mixed at a mass ratio of 1:2-4.

4. The method for preparing ginseng-derived nanoparticle-loaded drug according to any one of claims 1 or 2, characterized in that: The nanoparticles loaded with the drug have a particle size of 150-200 nm, a surface potential of -25 to -30 mV, and a bilayer film structure.

5. The application of the nanoparticle-loaded drug as described in claim 4 in the preparation of a liver injury repair drug, characterized in that: Ginseng exosomes are used to improve the oral bioavailability of drugs and deliver them to the liver. The anti-inflammatory and antioxidant activities of the ginseng exosomes, together with the loaded drug, reduce the ROS level in hepatocytes, while inhibiting hepatocyte apoptosis and enhancing cell viability.

6. The application of the nanoparticle-loaded drug according to claim 5 in the preparation of liver injury repair drugs, characterized in that: The drug-loaded nanoparticles repair the intestinal barrier by regulating the gut microbiota, thereby improving the liver microenvironment and achieving bidirectional regulation between the liver and gut.

7. The application of the nanoparticle-loaded drug as described in claim 4 in the preparation of an anti-liver fibrosis drug, characterized in that: It is used to inhibit the expression of fibrosis markers Vimentin and α-SMA in hepatic stellate cells, while also reducing collagen deposition and reversing CCl4-induced pathological features of mouse liver fibrosis.

8. The application of the nanoparticle-loaded drug according to claim 7 in the preparation of anti-liver fibrosis drugs, characterized in that: The drug-loaded nanoparticles restore liver function by reducing serum AST / ALT levels.

9. The application of the nanoparticle-loaded drug as described in claim 4 in the preparation of intestinal barrier repair drugs, characterized in that: The drug-loaded nanoparticles are used to repair the structural integrity of intestinal villi and upregulate the expression of intestinal tight junction proteins ZO-1 and Occludin.

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