Pharmaceutical composition for treating non-alcoholic fatty liver as well as preparation method and application thereof

Abstract

The invention discloses a pharmaceutical composition for treating non-alcoholic fatty liver as well as a preparation method and application thereof, and relates to the technical field of biological medicines. The invention discloses a pharmaceutical composition for treating non-alcoholic fatty liver disease. The pharmaceutical composition is prepared from a curdione / beta-cyclodextrin inclusion compound, glutaraldehyde cross-linked ganoderma lucidum polysaccharide and salidroside. According to the pharmaceutical composition developed by the invention, through the synergistic effect of curdione, ganoderan and salidroside, lipid metabolism disorder of a non-alcoholic fatty liver model is remarkably improved, liver cell injury is relieved, HE staining and oil red O staining prove that the pharmaceutical composition can reduce intrahepatic fat vacuoles and repair liver tissue structures, the effect of a medium dose group is optimal, and the effect of a low dose group is excellent. The effect is obviously better than that of each single component which is independently used, and a safe and efficient treatment scheme is provided for the non-alcoholic fatty liver disease.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a pharmaceutical composition for treating non-alcoholic fatty liver disease, its preparation method, and its application. Background Technology

[0002] Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease worldwide. Its pathological mechanisms involve multiple aspects such as lipid metabolism disorders, oxidative stress, chronic inflammation, and gut microbiota dysbiosis. If not intervened in time, it can progress to liver fibrosis, cirrhosis, and even liver cancer, seriously threatening human health.

[0003] Currently, the clinical treatment of non-alcoholic fatty liver disease is still based on lifestyle interventions, but patient compliance is poor and long-term effects are limited. Chemotherapy has problems such as hepatotoxicity and single target, and there are no approved specific drugs. Therefore, there is an urgent need to develop new drugs that are safe, effective, and have synergistic effects on multiple targets.

[0004] Traditional Chinese medicine (TCM) has advantages in the treatment of chronic metabolic diseases due to its multi-component, multi-target, and low-toxicity properties. Its active ingredients exhibit potential in combating non-alcoholic fatty liver disease (NAFLD) through mechanisms such as regulating metabolic pathways, inhibiting inflammatory responses, and improving oxidative damage. Therefore, the inventors believe that how to screen, construct, and build an active TCM composition for the treatment of NAFLD is a pressing technical problem that needs to be solved in this field.

[0005] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0006] To address the aforementioned technical problems, embodiments of the present invention provide a pharmaceutical composition for treating non-alcoholic fatty liver disease, its preparation method, and its application, thereby resolving the issues raised in the background section.

[0007] The present invention provides the following technical solution: a pharmaceutical composition for treating non-alcoholic fatty liver disease, comprising: turmeric dione / β-cyclodextrin inclusion complex, glutaraldehyde crosslinked Ganoderma lucidum polysaccharide and rhodioloside.

[0008] It should be noted that the purpose of β-cyclodextrin inclusion modification of turmeric dione is to address the fact that turmeric dione contains hydrophobic groups in its molecular structure, resulting in poor water solubility and difficulty in dissolving and absorbing it in vivo, directly affecting its bioavailability. Hydroxypropyl-β-cyclodextrin is a cyclodextrin derivative modified with hydroxypropyl, possessing a hydrophilic shell and a hydrophobic cavity. The hydrophobic cavity can form a stable inclusion complex with the lipid-soluble turmeric dione through van der Waals forces and hydrophobic interactions. β-cyclodextrin inclusion modification of turmeric dione significantly improves its overall water solubility, solving the problem of its poor solubility and absorption, making it easier to be absorbed through the gastrointestinal tract and reach the liver to exert its effects.

[0009] The purpose of cross-linking Ganoderma lucidum polysaccharides with glutaraldehyde is that Ganoderma lucidum polysaccharide molecules are rich in hydroxyl groups, which are easily degraded by intestinal flora or glycosidases in vivo and are difficult to exert their effects continuously. Glutaraldehyde, as a bifunctional cross-linking agent, can undergo a condensation reaction with the hydroxyl groups in Ganoderma lucidum polysaccharide molecules to form a stable covalent cross-linked network macromolecule, which significantly enhances its resistance to enzymatic degradation. Uncrosslinked Ganoderma lucidum polysaccharides are too water-soluble and are easily lost quickly with body fluids; the network structure formed after crosslinking can reduce water solubility, increase its retention time in the gastrointestinal tract, and improve intestinal absorption efficiency; at the same time, crosslinked Ganoderma lucidum polysaccharides have large molecular weight and stable structure, and can be absorbed through the intestinal lymphatic system, avoiding the first-pass effect of the liver, and further improving bioavailability.

[0010] Preferably, by mass parts, it includes: 1-3 parts of curcumindione / β-cyclodextrin inclusion complex, 2-5 parts of glutaraldehyde-crosslinked Ganoderma lucidum polysaccharide, and 1-3 parts of rhodioloside.

[0011] Preferably, by mass parts, it includes: 2 parts of curcumindione / β-cyclodextrin inclusion complex, 3 parts of glutaraldehyde cross-linked Ganoderma lucidum polysaccharide, and 2 parts of rhodioloside.

[0012] Preferably, in the curcumin / β-cyclodextrin inclusion complex, the mass ratio of curcumin to β-cyclodextrin is 1:3-8; and the cyclodextrin is hydroxypropyl-β-cyclodextrin.

[0013] Preferably, the pharmaceutical excipients are also included, including microcrystalline cellulose, low-substituted hydroxypropyl cellulose, crospovidone, and magnesium stearate; the formulation of the pharmaceutical excipients facilitates the formulation compression of the pharmaceutical composition.

[0014] A method for preparing a pharmaceutical composition for treating non-alcoholic fatty liver disease as described above includes the following steps: S61: Curcuma dione and β-cyclodextrin are mixed and dissolved in deionized water; then curcuma dione is incorporated into cyclodextrin to obtain curcuma dione / β-cyclodextrin inclusion complex; S62: Ganoderma lucidum polysaccharide is dissolved in deionized water, and glutaraldehyde is added to carry out a cross-linking reaction to obtain glutaraldehyde-cross-linked Ganoderma lucidum polysaccharide; S63: Curcuma dione / β-cyclodextrin inclusion complex, glutaraldehyde cross-linked Ganoderma lucidum polysaccharide and rhodioloside are mixed evenly to obtain a pharmaceutical composition for treating non-alcoholic fatty liver disease.

[0015] Preferably, the specific steps for preparing the turmeric dione / β-cyclodextrin inclusion complex in step S61 include: Dissolve hydroxypropyl-β-cyclodextrin in deionized water and stir in a water bath at 60-70℃ until completely dissolved to prepare a cyclodextrin solution with a mass fraction of 20-40%. Add turmeric dione to β-cyclodextrin at a mass ratio of 1:3-8, and continue stirring for 2-4 hours to form an inclusion solution. Refrigerate the inclusion solution for 12-24 hours, filter, and freeze-dry to obtain the turmeric dione / β-cyclodextrin inclusion complex.

[0016] Preferably, the specific steps in step S62 for preparing glutaraldehyde-crosslinked Ganoderma lucidum polysaccharides include: Dissolve Ganoderma lucidum polysaccharides in deionized water to prepare a first solution with a mass fraction of 5-10%. Glutaraldehyde was added to the first solution; the mass ratio of glutaraldehyde to Ganoderma lucidum polysaccharide was 0.05-0.2:1. The reaction was stirred for 1-2 hours at 30-40℃ and pH 5-6; the pH of the solution was adjusted using an acetate-sodium acetate buffer solution. Ethanol was added to precipitate the product, which was then collected by centrifugation, washed, and freeze-dried to obtain glutaraldehyde-crosslinked Ganoderma lucidum polysaccharide.

[0017] Preferably, the specific steps in step S63 for preparing the pharmaceutical composition for treating non-alcoholic fatty liver disease include: By weight, 1-3 parts of turmeric dione / β-cyclodextrin inclusion complex, 2-5 parts of glutaraldehyde-crosslinked Ganoderma lucidum polysaccharide, and 1-3 parts of rhodioloside are mixed to obtain a pharmaceutical composition for treating non-alcoholic fatty liver disease.

[0018] Preferably, the dosage form of the pharmaceutical composition for treating non-alcoholic fatty liver disease is sustained-release microcapsules; the method for preparing sustained-release microcapsules includes the following steps: Mix 1-3 parts of turmeric dione / β-cyclodextrin inclusion complex, 2-5 parts of glutaraldehyde crosslinked Ganoderma lucidum polysaccharide and 1-3 parts of rhodioloside evenly, add to 1% sodium alginate solution by mass, and stir evenly to obtain aqueous phase; The aqueous phase was slowly added dropwise to the oil phase containing 2% calcium chloride by mass, and stirred to form a W / O emulsion; wherein the oil phase consisted of liquid paraffin and Span-80; Continue adding 1% (w / w) chitosan solution, and allow the cross-linking reaction to proceed for 30-60 minutes. Centrifuge to collect the microcapsules, wash, and freeze-dry to obtain sustained-release microcapsules.

[0019] The use of a pharmaceutical composition for treating non-alcoholic fatty liver disease as described above in the treatment of non-alcoholic fatty liver disease.

[0020] The pharmaceutical composition for treating non-alcoholic fatty liver disease provided in this invention has the following beneficial effects: The pharmaceutical composition developed in this invention, through the synergistic effect of curcuminidone, Ganoderma lucidum polysaccharide and rhodioloside, significantly improves lipid metabolism disorder in a non-alcoholic fatty liver disease model (reducing serum TG and liver tissue TC) and alleviates hepatocyte damage (reducing ALT levels). HE staining and Oil Red O staining have confirmed that it can reduce intrahepatic fat vacuoles and repair liver tissue structure. The medium-dose group has the best effect and the effect is significantly better than that of each single component used alone, providing a safe and efficient treatment option for non-alcoholic fatty liver disease. Attached Figure Description

[0021] Figure 1 HE staining images of liver tissue from mice in the control group and model group; Where KB is the blank group; Model is the model group; Figure 2 HE staining image of liver tissue from mice in the control group; Among them, Cur is the curcuminone group; Gan is the Ganoderma lucidum polysaccharide group; Sal is the rhodioloside group; Figure 3 HE staining image of liver tissue from mice in the drug treatment group of this invention; Wherein, L is the low-dose group; M is the medium-dose group; and H is the high-dose group; Figure 4 Oil Red O staining images of liver tissue from mice in the control group and model group; Where KB is the blank group; Model is the model group; Figure 5 Oil Red O staining images of liver tissue from mice in the comparison group; Among them, Cur is the curcuminone group; Gan is the Ganoderma lucidum polysaccharide group; Sal is the rhodioloside group; Figure 6 This is an image of Oil Red O staining of liver tissue from mice in the drug treatment group of this invention; Among them, L is the low-dose group; M is the medium-dose group; and H is the high-dose group. Detailed Implementation

[0022] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. 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.

[0023] To address the problems mentioned in the background section, this invention provides a pharmaceutical composition for treating non-alcoholic fatty liver disease, its preparation method, and its application, thereby solving the aforementioned technical problems. The specific implementation method is as follows: Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0024] Example 1: The effect of the pharmaceutical composition on improving FFA-induced steatosis in LO2 cells (a) Experimental materials Cell line: LO2 cells (human normal liver cell line) were purchased from the Cell Bank of the Chinese Academy of Sciences and cultured in DMEM medium containing 10% fetal bovine serum.

[0025] The formulations of the drug of this invention for low, medium, and high dose groups are as follows: Low-dose formulation: 1 part curcumindione / β-cyclodextrin inclusion complex, 2 parts glutaraldehyde cross-linked Ganoderma lucidum polysaccharide, and 1 part rhodioloside; Medium-dose formulation: 2 parts of curcumindione / β-cyclodextrin inclusion complex, 3 parts of glutaraldehyde cross-linked Ganoderma lucidum polysaccharide, and 2 parts of rhodioloside; High-dose formulation: 3 parts of curcumindione / β-cyclodextrin inclusion complex, 5 parts of glutaraldehyde cross-linked Ganoderma lucidum polysaccharide, and 3 parts of rhodioloside; Reagents: Palmitic acid (PA) and oleic acid (OA) were purchased from Sigma and used as free fatty acids (FFA, modeling reagent); Oil Red O staining kit and cell TG detection kit were purchased from Beyotime.

[0026] The preparation methods for the low, medium, and high dose groups of drugs include the following steps: Curcuma dione was mixed with β-cyclodextrin and dissolved in deionized water; then, curcuma dione was incorporated into the cyclodextrin to obtain a curcuma dione / β-cyclodextrin inclusion complex. Ganoderma lucidum polysaccharides were dissolved in deionized water, and glutaraldehyde was added to carry out a cross-linking reaction to obtain glutaraldehyde-cross-linked Ganoderma lucidum polysaccharides. Curcuma dione / β-cyclodextrin inclusion complex, glutaraldehyde-crosslinked Ganoderma lucidum polysaccharide, and rhodioloside were mixed evenly to obtain a pharmaceutical composition for treating non-alcoholic fatty liver disease.

[0027] (II) Experimental Methods Cell model establishment: LO2 cells were seeded in 6-well plates (5 × 10⁻⁶ cells / well). 5 Cells were cultured for 24 hours and then treated with medium containing 0.5 mmol / L LFFA (PA:OA=1:2) for 24 hours to induce fatty degeneration.

[0028] Group processing: Blank control group: normal culture medium; Model group: 0.5 mmol / LFFA; Curcuma dione / β-cyclodextrin inclusion complex comparison group: 0.5 mmol / LFFA + 5 μg / mL of curcuma dione / β-cyclodextrin inclusion complex; Glutaraldehyde-crosslinked Ganoderma lucidum polysaccharide control group: 0.5 mmol / LFFA + glutaraldehyde-crosslinked Ganoderma lucidum polysaccharide 5 μg / mL; Rhodioloside control group: 0.5 mmol / LFFA + 5 μg / mL rhodioloside; Low-dose group: 0.5 mmol / LFFA + 5 μg / mL of the drug composition of the low-dose group formulation; Medium-dose group: 0.5 mmol / LFFA + 5 μg / mL of the drug composition of the medium-dose group formulation; High-dose group: 0.5 mmol / LFFA + 5 μg / mL of the drug composition from the high-dose group formulation; The indicators were measured 24 hours after treatment in each group.

[0029] Testing indicators: Oil Red O staining: After cell fixation, staining was performed, and lipid droplet distribution was observed under a microscope. After isopropanol extraction, absorbance at 510 nm was measured (to quantify lipid droplet content). Intracellular TG (triglyceride) content: Detected according to the kit instructions, expressed as mmol / g protein.

[0030] (III) Experimental Results Observation of lipid droplet morphology: A large number of red lipid droplets appeared in the cells of the model group, indicating that FFA treatment induced fatty degeneration, which is a manifestation of intracellular fat accumulation.

[0031] The number of lipid droplets decreased in all three groups (low, medium, and high doses), with the medium dose showing the most significant reduction (P<0.01). This indicates that the drug composition helps alleviate fatty degeneration caused by FFA, and that the medium dose has the best effect.

[0032] The number of intracellular lipid droplets in the turmeric dione / β-cyclodextrin inclusion complex, glutaraldehyde cross-linked Ganoderma lucidum polysaccharide, and rhodioloside comparison groups were all reduced to some extent compared with the model group, but the degree of reduction was significantly weaker than that in the low, medium, and high dose groups. This indicates that the effect of a single drug component on improving cellular steatosis is limited and far less than the synergistic effect of the drug composition of the present invention.

[0033] Quantitative analysis: The absorbance of lipid droplets in the model group (0.85±0.07) was significantly higher than that in the control group (0.21±0.03, P<0.01); the significantly higher absorbance of lipid droplets in the model group indicates that FFA-induced fatty degeneration leads to the accumulation of lipid droplets.

[0034] The absorbance of the medium-dose group (0.32±0.04) was significantly lower than that of the low-dose group (0.58±0.05) and the high-dose group (0.45±0.06, P<0.05), indicating that the medium-dose drug had the best effect and could effectively reduce fatty degeneration.

[0035] Intracellular TG (triglyceride) content: The model group (1.92±0.18 mmol / g protein) was significantly increased, while the medium-dose group (0.76±0.09 mmol / g protein) showed the most significant decrease (P<0.01); indicating that the medium-dose drug can effectively reduce intracellular TG content and alleviate fatty degeneration.

[0036] Among them, the absorbance of the curcumin / β-cyclodextrin inclusion complex was 0.72±0.06, the absorbance of the glutaraldehyde cross-linked Ganoderma lucidum polysaccharide complex was 0.75±0.07, and the absorbance of the rhodioloside complex was 0.73±0.06; although the absorbance of all three groups was significantly lower than that of the low, medium and high dose groups. The TG content of the curcumin dione / β-cyclodextrin inclusion complex was 1.65±0.15 mmol / g protein in the control group, 1.72±0.16 mmol / g protein in the glutaraldehyde cross-linked Ganoderma lucidum polysaccharide control group, and 1.68±0.15 mmol / g protein in the rhodioloside control group.

[0037] The above results further confirm that although the single component has a certain ameliorative effect on FFA-induced cellular steatosis, its effect is significantly weaker than that of the pharmaceutical composition of the present invention.

[0038] Example 2: Therapeutic effect of the pharmaceutical composition on NAFLD mice induced by a high-fat diet. (a) Experimental materials Animals: 40 male C57BL / 6 mice (20±2g) aged 8 weeks.

[0039] The drug and feed of the present invention: The drug of the present invention is the same as the drug composition of Example 1; high-fat feed (60% basal feed + 20% lard + 10% sucrose + 2% cholesterol); high-fat feed (HFD).

[0040] (II) Experimental Methods Grouping and Modeling: Mice were randomly divided into 5 groups (n=8); Control group: Normal diet + saline gavage; Model group: HFD + physiological saline 20 mg / kg by gavage; Curcuma dione / β-cyclodextrin inclusion complex comparison group: HFD + curcuma dione / β-cyclodextrin inclusion complex 20 mg / kg by gavage; Glutaraldehyde-crosslinked Ganoderma lucidum polysaccharide control group: HFD + glutaraldehyde-crosslinked Ganoderma lucidum polysaccharide 20 mg / kg administered by gavage; Rhodioloside control group: HFD + Rhodioloside 20 mg / kg by gavage; Low-dose group: HFD + low-dose group formulation 20 mg / kg drug composition was administered by gavage; Medium-dose group: HFD+ medium-dose group formulation 20 mg / kg drug composition was administered by gavage; High-dose group: HFD + high-dose group formulation 20 mg / kg drug composition administered by gavage; Administer by gavage daily for 8 consecutive weeks at a drug concentration of 2 mg / mL.

[0041] The preparation methods for the low, medium, and high dose groups are the same as those in Example 1.

[0042] Sample collection: After fasting for 12 hours following the last administration, the animals were weighed, and blood was drawn from the eyeballs (to measure serum TG, TC, and ALT). The animals were then euthanized and their livers were harvested (the liver index was calculated, and liver tissue TG, TC, HE, and Oil Red O staining were performed).

[0043] (III) Serum and Liver Tissue Indicator Results The results of serum and liver tissue indicators are shown in Table 1: Group Blood TG (mmol / L) Total cholesterol (TC) in liver tissue (mmol / g) Serum ALT (U / L) control group 1.12±0.11 2.25±0.18 23.5±2.8 Model group 3.15±0.29## 5.92±0.41## 75.6±5.2## Curcuma dione / β-cyclodextrin inclusion complex comparison group 2.68±0.23* 4.85±0.36* 61.2±4.9* Glutaraldehyde cross-linked Ganoderma lucidum polysaccharide control group 2.82±0.25* 5.12±0.38* 65.4±5.1* Rhodiola rosea glycosides comparison group 2.75±0.24* 4.98±0.37* 63.5±5.0* low-dose group 2.16±0.21* 4.18±0.32* 52.3±4.5* medium dose group 1.45±0.15** 2.86±0.25** 31.2±3.1** High-dose group 1.89±0.17* 3.52±0.28* 40.5±3.8* Note: Compared with the control group, ##P<0.01; compared with the model group, P<0.05, **P<0.01; the medium-dose group showed the most significant decrease in serum and liver tissue lipid and ALT levels (P<0.01).

[0044] The results of Table 1, "Serium and Liver Tissue Indicators," are analyzed as follows: (1) Changes in indicators in the model group compared to the control group Serum triglycerides (TG): The model group (3.15±0.29 mmol / L) was significantly higher than that of the control group (1.12±0.11 mmol / L), and the difference was extremely significant (##P<0.01), indicating that the high-fat diet-induced non-alcoholic fatty liver model mice showed significant increases in serum triglycerides and lipid metabolism disorders.

[0045] Total cholesterol (TC) in liver tissue: The model group (5.92±0.41 mmol / g) was significantly higher than that in the control group (2.25±0.18 mmol / g), with a highly significant difference (##P<0.01), indicating that a large amount of total cholesterol accumulated in the liver of mice in the model group, which is consistent with the pathological characteristics of fatty liver.

[0046] Serum ALT (alanine aminotransferase): The model group (75.6±5.2U / L) was significantly higher than that of the control group (23.5±2.8U / L), with an extremely significant difference (##P<0.01), suggesting that a high-fat diet leads to liver cell damage and significant abnormalities in liver function in mice (ALT is an important indicator of liver cell damage).

[0047] The above results indicate that a high-fat diet successfully established a non-alcoholic fatty liver disease model, and the model group mice showed obvious lipid metabolism abnormalities and hepatocyte damage.

[0048] (2) Changes in indicators in each treatment group compared with the model group In the low-dose group, serum TG (2.16±0.21 mmol / L), liver tissue TC (4.18±0.32 mmol / g), and serum ALT (52.3±4.5 U / L) were significantly lower than those in the model group, with statistically significant differences (*P<0.05). This indicates that the low-dose drug composition can improve lipid metabolism disorders and hepatocellular damage to a certain extent, but the effect is moderate.

[0049] In the medium-dose group, serum TG (1.45±0.15 mmol / L), liver tissue TC (2.86±0.25 mmol / g), and serum ALT (31.2±3.1 U / L) were all significantly lower than those in the model group, and the differences were extremely significant (**P<0.01). Compared with the low-dose and high-dose groups, the medium-dose group showed the greatest reduction in the three indicators (e.g., serum TG was close to the control group level, liver tissue TC was only slightly higher than the control group, and ALT decreased significantly), indicating that the medium-dose drug composition had the most significant effect on improving lipid metabolism disorders and repairing hepatocellular damage.

[0050] In the high-dose group, serum TG (1.89±0.17 mmol / L), liver tissue TC (3.52±0.28 mmol / g), and serum ALT (40.5±3.8 U / L) were significantly lower than those in the model group (*P<0.05), but all indicators were higher than those in the medium-dose group. This indicates that the high-dose drug combination is more effective than the low-dose group, but not as effective as the medium-dose group, and there is a certain dose-response difference (it is not that the higher the dose, the better the effect).

[0051] (4) Changes in indicators of each comparison group compared with the model group and the drug treatment group The serum TG, liver tissue TC, and serum ALT levels in each of the single-component comparison groups (turmeric dione / β-cyclodextrin inclusion complex comparison group, glutaraldehyde cross-linked Ganoderma lucidum polysaccharide comparison group, and rhodioloside comparison group) were significantly lower than those in the model group (*P<0.05), indicating that the single components have a certain ameliorative effect on lipid metabolism disorder and hepatocyte damage in the NAFLD model, but the ameliorative effect is significantly weaker than that of each dose group of the drug composition.

[0052] (4) Conclusion: The results in Table 1 confirm that the pharmaceutical composition of the present invention has an ameliorative effect on lipid metabolism disorder and hepatocyte damage in the non-alcoholic fatty liver model. Among them, the medium dose group (preferred formula) has the most significant effect, with the largest reduction in serum TG, liver tissue TC and serum ALT levels, which are close to the levels of the normal control group. This reflects the synergistic effect of each component in the composition at this ratio, and provides the optimal dosage reference for the treatment of non-alcoholic fatty liver.

[0053] (iv) Results of HE staining of the liver Figure 1 The results of H&E staining of the liver showed that the liver tissue of the control group mice was uniformly stained, the hepatocyte cords were arranged regularly, the sinusoids were clear, and there were no fat vacuoles or inflammatory cell infiltration.

[0054] After 8 weeks of high-fat diet induction, the liver tissue of mice in the model group showed disordered lobular structure, obvious swelling of hepatocytes, a large number of fat vacuoles of varying sizes, significant ballooning degeneration of hepatocytes, scattered punctate and focal necrosis in the lobules, and enlargement of portal areas accompanied by a large number of lymphocytes and inflammatory cells infiltrating.

[0055] Figure 3 Liver H&E staining results showed that after low-dose drug intervention, the lobular structure of mouse liver tissue was slightly improved, the number of fat vacuoles was reduced compared with the model group, but there were still many scattered vacuoles, and the inflammatory cell infiltration in the portal area was reduced but not completely eliminated.

[0056] After intervention with the medium-dose group, the lobular structure of the mouse liver tissue was basically intact, the hepatocyte cords were arranged more neatly, the fat vacuoles were significantly reduced, the ballooning degeneration of hepatocytes basically disappeared, and the inflammatory cell infiltration in the portal area was significantly reduced, approaching the morphology of normal liver tissue.

[0057] After intervention with high-dose drugs, the number of fat vacuoles in the liver tissue of mice was reduced compared with that in the model group, and mild inflammatory cell infiltration was still present in local areas. The improvement effect was not as good as that in the medium-dose group.

[0058] Figure 2 Liver H&E staining results showed that the three single-component comparison groups all had a certain improvement effect on liver steatosis and inflammatory infiltration, but the effect was significantly weaker than that of the low-dose drug combination group. Specifically, the reduction of fat vacuoles, the degree of lobular structure repair and the efficiency of inflammatory cell clearance were all inferior to those of the drug combination group, further confirming that the synergistic effect of turmeric dione, Ganoderma lucidum polysaccharide and rhodioloside on repairing pathological damage in liver tissue was better.

[0059] (v) Results of liver oil red O staining Figure 4Oil Red O staining of the liver showed that the control group hepatocytes had almost no orange-red lipid droplets in their cytoplasm; the model group hepatocytes were filled with a large number of dense orange-red lipid droplets, which were obviously fused and occupied most of the cell space.

[0060] Figure 6 Oil Red O staining of the liver showed that the number of lipid droplets decreased in all treatment groups. Specifically, the number of lipid droplets in the low-dose group was significantly reduced compared to the model group, but there were still many scattered lipid droplets. The number of lipid droplets in the medium-dose group was significantly reduced, with only a small number of scattered micro-lipid droplets observed locally, and the distribution was sparse. The number of lipid droplets in the high-dose group was significantly reduced compared to the model group, but more than that in the medium-dose group.

[0061] Figure 5 The results of liver oil red O staining showed that although the three single-component comparison groups could reduce intrahepatic lipid droplet deposition, the effect was significantly weaker than that of each dose group of the drug composition. Moreover, the reduction in lipid droplets, their size distribution, and staining intensity were all inferior to those of the medium-dose group, further confirming that the synergistic effect of the multiple components in the composition on the regulation of intrahepatic lipid metabolism was more significant.

[0062] The above Figures 1-6 The results showed that the medium-dose group had the most significant effect on improving lipid deposition in hepatocytes. Although the low-dose and high-dose groups had some effect, the effect was not as good as that of the medium-dose group. This further confirms that the drug composition has the best regulatory effect on lipid metabolism disorder in non-alcoholic fatty liver disease at the medium dose.

[0063] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A pharmaceutical composition for treating non-alcoholic fatty liver disease, characterized in that, include: Curcumin dione / β-cyclodextrin inclusion complex, glutaraldehyde cross-linked Ganoderma lucidum polysaccharide and rhodioloside.

2. The pharmaceutical composition for treating non-alcoholic fatty liver disease according to claim 1, characterized in that, By mass fractions, it includes: 1-3 parts of curcumindione / β-cyclodextrin inclusion complex, 2-5 parts of glutaraldehyde-crosslinked Ganoderma lucidum polysaccharide, and 1-3 parts of rhodioloside.

3. The pharmaceutical composition for treating non-alcoholic fatty liver disease according to claim 1, characterized in that, By mass fraction, it includes: 2 parts of curcumindione / β-cyclodextrin inclusion complex, 3 parts of glutaraldehyde cross-linked Ganoderma lucidum polysaccharide, and 2 parts of rhodioloside.

4. The pharmaceutical composition for treating non-alcoholic fatty liver disease according to claim 1, characterized in that, In the curcumin / β-cyclodextrin inclusion complex, the mass ratio of curcumin to β-cyclodextrin is 1:3-8; the cyclodextrin is hydroxypropyl-β-cyclodextrin.

5. The pharmaceutical composition for treating non-alcoholic fatty liver disease according to claim 1, characterized in that, It also includes pharmaceutical excipients, including microcrystalline cellulose, low-substituted hydroxypropyl cellulose, crospovidone, and magnesium stearate.

6. A method for preparing a pharmaceutical composition for treating non-alcoholic fatty liver disease as described in claim 1, characterized in that, Includes the following steps: S61: Curcuma dione and β-cyclodextrin are mixed and dissolved in deionized water; then curcuma dione is included in cyclodextrin to obtain curcuma dione / β-cyclodextrin inclusion complex; S62: Ganoderma lucidum polysaccharide is dissolved in deionized water, and glutaraldehyde is added to carry out a cross-linking reaction to obtain glutaraldehyde-cross-linked Ganoderma lucidum polysaccharide; S63: Curcuma dione / β-cyclodextrin inclusion complex, glutaraldehyde cross-linked Ganoderma lucidum polysaccharide and rhodioloside are mixed evenly to obtain a pharmaceutical composition for treating non-alcoholic fatty liver disease.

7. The method for preparing the pharmaceutical composition for treating non-alcoholic fatty liver disease according to claim 6, characterized in that, The specific steps in step S61 for preparing the turmeric dione / β-cyclodextrin inclusion complex include: Dissolve hydroxypropyl-β-cyclodextrin in deionized water and stir in a water bath at 60-70℃ until completely dissolved to prepare a cyclodextrin solution with a mass fraction of 20-40%. Add turmeric dione to β-cyclodextrin at a mass ratio of 1:3-8, and continue stirring for 2-4 hours to form an inclusion solution. Refrigerate the inclusion solution for 12-24 hours, filter, and freeze-dry to obtain the turmeric dione / β-cyclodextrin inclusion complex.

8. The method for preparing the pharmaceutical composition for treating non-alcoholic fatty liver disease according to claim 6, characterized in that, The specific steps in step S62 for preparing glutaraldehyde-crosslinked Ganoderma lucidum polysaccharides include: Dissolve Ganoderma lucidum polysaccharides in deionized water to prepare a first solution with a mass fraction of 5-10%. Glutaraldehyde was added to the first solution; the mass ratio of glutaraldehyde to Ganoderma lucidum polysaccharide was 0.05-0.2:

1. The reaction was stirred for 1-2 hours at 30-40℃ and pH 5-6; the pH of the solution was adjusted using an acetate-sodium acetate buffer solution. Ethanol was added to precipitate the product, which was then collected by centrifugation, washed, and freeze-dried to obtain glutaraldehyde-crosslinked Ganoderma lucidum polysaccharide.

9. The method for preparing the pharmaceutical composition for treating non-alcoholic fatty liver disease according to claim 6, characterized in that, The specific steps in step S63 for preparing the pharmaceutical composition for treating non-alcoholic fatty liver disease include: By weight, 1-3 parts of turmeric dione / β-cyclodextrin inclusion complex, 2-5 parts of glutaraldehyde-crosslinked Ganoderma lucidum polysaccharide, and 1-3 parts of rhodioloside are mixed to obtain a pharmaceutical composition for treating non-alcoholic fatty liver disease.

10. The use of the pharmaceutical composition for treating non-alcoholic fatty liver disease as described in claim 1 in the treatment of non-alcoholic fatty liver disease.

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