Application of hyperoside and salvianolic acid B in preparation of medicine for treating NAFLD

Abstract

The present application relates to the application of hyperoside and salvianolic acid B in the preparation of a drug for treating non-alcoholic fatty liver disease, and belongs to the technical field of biological medicine. The hyperoside and salvianolic acid B have a synergistic effect in the preparation of a drug for treating non-alcoholic fatty liver disease when used in combination in a required amount, and the combination of the two drugs can enhance the curative effect and reduce side effects, which is more effective than the use of a single drug, provides a new idea for treating non-alcoholic fatty liver disease, and has important significance and wide application prospect.

Description

Technical Field

[0001] This invention relates to the application of hyperoside and salvianolic acid B in the preparation of a drug for the treatment of NAFLD, belonging to the field of biomedical technology. Background Technology

[0002] Non-alcoholic fatty liver disease (NAFLD) is a form of hepatic steatosis caused by non-alcoholic factors and is a typical liver manifestation of metabolic syndrome. The incidence of NAFLD is increasing year by year and can further develop into steatohepatitis, cirrhosis, and even liver cancer, making it one of the important causes of chronic liver disease.

[0003] Current treatment strategies primarily focus on lifestyle interventions. With only resmetirom approved by the FDA in 2024 for the treatment of NAFLD, existing treatment options are extremely limited. Therefore, there is an urgent need to develop novel, highly effective, and low-toxicity therapeutic drugs.

[0004] Hyperoside (Hyp) is a flavonol glycoside. Existing research shows that hyperoside can significantly reduce the content of free bile acids in the liver of rats by activating the hepatic FXR protein, thereby alleviating the toxic effects of bile acids on the liver. It also promotes the excretion of lipids and bile acids, accelerates fatty acid oxidation, and inhibits lipid synthesis. Its pharmacological effects suggest that its application in NAFLD may be a new area. However, further research has revealed that hyperoside suffers from unstable efficacy and insignificant anti-inflammatory effects, limiting its application.

[0005] Salvianolic acid B (Sal B) is a hydrophilic active ingredient extracted from Salvia miltiorrhiza. It is a natural polyphenol compound with pharmacological effects including antitumor, antithrombotic, antifibrotic, anti-inflammatory, and antioxidant properties. It also offers some protection to the heart, liver, brain nerves, and blood vessels. Furthermore, current research has shown that certain concentrations of Sal B can improve non-alcoholic fatty liver disease, particularly in regulating lipids. Mechanistic studies have revealed that Salvianolic acid B can inhibit the expression of intestinal LIMA1 protein. LIMA1 protein has been shown to play a role in the transport of the cholesterol transporter NPC1L1 from endocytosis to the cell membrane, thereby affecting cholesterol uptake.

[0006] To date, hyperoside combined with salvianolic acid B has not been used in the treatment of non-alcoholic fatty liver disease. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides an application of a drug for treating NAFLD prepared by combining hyperoside and salvianolic acid B.

[0008] The technical solution used in this invention is as follows:

[0009] Application of drugs for the treatment and / or prevention of NAFLD prepared by combining hyperoside and salvianolic acid B.

[0010] According to a preferred embodiment of the present invention, when administered at the cellular level, the concentration of salvianolic acid B is 2.5~5 μM, the concentration of hyperoside is 3.75~7.5 μM, and the molar ratio of salvianolic acid B to hyperoside is 2:3.

[0011] According to a preferred embodiment of the present invention, when administered at the animal level, the concentration of salvianolic acid B is 10 mg / kg and the concentration of hyperoside is 1.5 mg / kg.

[0012] A medicine for the prevention and / or treatment of NAFLD, wherein the active ingredients of the medicine include hyperoside and salvianolic acid B.

[0013] According to a preferred embodiment of the present invention, when administered at the cellular level, the concentration of salvianolic acid B is 2.5~5 μM, the concentration of hyperoside is 3.75~7.5 μM, and the molar ratio of salvianolic acid B to hyperoside is 2:3.

[0014] According to a preferred embodiment of the present invention, when administered at the animal level, the concentration of salvianolic acid B is 10 mg / kg and the concentration of hyperoside is 1.5 mg / kg.

[0015] According to a preferred embodiment of the invention, the drug contains one or more pharmaceutically acceptable carriers or excipients.

[0016] More preferably, the excipient is at least one of a sustained-release agent, excipient, filler, binder, wetting agent, disintegrant, absorption promoter, surfactant, or lubricant.

[0017] According to a preferred embodiment of the present invention, the dosage form of the drug is tablet, pill, capsule, oral liquid or injection.

[0018] Beneficial effects:

[0019] When hyperoside and salvianolic acid B are used in combination at the required therapeutic dose, they have a synergistic effect in the preparation of drugs for the treatment of non-alcoholic fatty liver disease. The combined use of the two drugs can enhance the efficacy and reduce the side effects, which is greater than the effect of using the drugs alone. This provides a new approach to the treatment of non-alcoholic fatty liver disease and has important significance and broad application prospects. Attached Figure Description

[0020] Figure 1 A bar chart showing the results of TG content measurement in cells of different drug administration groups;

[0021] Figure 2The following are some of the results of the Chou-Talalay joint index analysis; where A is a scatter plot of the joint index and Fa value, and B is a curve showing the relationship between the logarithm of the joint index and Fa value.

[0022] Figure 3 The bar chart shows the results of TG content measurement in mouse liver; * indicates p < 0.05 compared with the model group; **** indicates p < 0.0001 compared with the model group.

[0023] Figure 4 Image showing the results of H&E staining in mouse liver;

[0024] Figure 5 Image showing the results of Oil Red O staining of mouse liver;

[0025] Figure 6 The bar chart shows the results of AST activity assay in mouse serum; * indicates p < 0.05 compared to the model group. Detailed Implementation

[0026] The technical solution of the present invention will be further described below with reference to the embodiments, but the scope of protection of the present invention is not limited thereto.

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; and the materials and reagents used are commercially available unless otherwise specified.

[0028] Unless otherwise specified, the pH of the PBS buffer used in the following experiments is 7.4.

[0029] Example 1

[0030] Cellular level experiments

[0031] 1. Drug preparation:

[0032] Preparation of hyperoside solution: Accurately weigh 5.57 mg Hyp, dissolve it in 100 μL of dimethyl sulfoxide (DMSO), and sonicate to obtain 120 mM hyperoside stock solution. After dispensing, store in a -20°C refrigerator.

[0033] Preparation of salvianolic acid B solution: Accurately weigh 5.75 mg SalB, dissolve in 100 μL DMSO, sonicate to dissolve, and obtain 80 mM salvianolic acid B stock solution. After dispensing, store in a -20℃ refrigerator.

[0034] Preparation of PA-d-BSA solution: (1) Preparation of 20% d-BSA solution: Preheat PBS buffer to 55℃, measure 0.6g BSA (bovine serum albumin), place it in a 10 mL centrifuge tube, add 3mL PBS buffer, do not mix or shake, directly place in a centrifuge at 12000rpm for 20 min, until BSA is completely dissolved, and obtain 3mL 20% d-BSA solution, which is a clear brownish-yellow sample. (2) Preparation of 20mM PA solution: Take 0.04g NaOH, dissolve it in 10 mL of pure water to prepare 10 mL of 0.1mol / L NaOH solution, take 0.01536g PA (palmitic acid) and add it to 3mL of NaOH solution, place it in a 75℃ water bath for full saponification for 30 min, and finally present a colorless, transparent, clear sample, and obtain 20 mM PA solution. Maintain the temperature and continue to the next step. (3) Add the insulated PA solution to the d-BSA solution to obtain 6 mL of 10 mM PA + 10% d-BSA solution, i.e., PA-d-BSA solution. Shake well and place in a 55℃ water bath for 30 min to aid dissolution, resulting in a clear brownish-yellow liquid. Dispense the liquid and store it in a -20℃ refrigerator.

[0035] Preparation of OA-d-BSA solution: (1) Preparation of 40% d-BSA solution: Preheat PBS buffer to 55℃, measure 1.2g BSA, place it in a 10 mL centrifuge tube, add 3mL PBS buffer, and then centrifuge directly in a centrifuge at 12000rpm for 20min until the BSA is completely dissolved, to obtain 3 mL of 40% d-BSA solution. (2) Preparation of 40 mM OA solution: Take 0.04g NaOH, dissolve it in 10 mL of pure water to prepare 10 mL of 0.1mol / L NaOH solution, add 38.08μL OA (oleic acid) to 3 mL of NaOH solution, and place it in a 75℃ water bath for 30 min to fully saponify. Finally, it becomes colorless, transparent and clear, to obtain 40 mM OA solution. Maintain the temperature and continue to the next step. (3) Quickly add the insulated OA solution to the d-BSA solution to obtain 6 mL of 20 mM OA + 20% d-BSA solution, i.e., OA-d-BSA solution. Shake well and place in a 55℃ water bath for 30 min to aid dissolution, resulting in a clear brownish-yellow liquid. Dispense the liquid and store it in a -20℃ refrigerator.

[0036] 2. Cell treatment

[0037] (1) Cell plating

[0038] AML-12 cells (purchased from Pronos) were cultured in AML-12 medium in a humidified cell culture incubator at 37°C and 5% CO2. AML-12 cells with 80%-90% confluence were prepared. After discarding the old medium, the cells were gently washed twice with PBS buffer. 2 mL of trypsin was added, and the cells were digested at 37°C for 1-2 minutes until they became rounded. Immediately, an equal volume of AML-12 medium was added to stop the digestion, and the cells were pipetted to form a single-cell suspension. After centrifugation at 1000 rpm for 5 minutes, the supernatant was discarded. The cells were resuspended in 1 mL of AML-12 medium and counted using trypan blue. The cell concentration was adjusted to 5 × 10⁶ cells / mL. 4 cells / mL. Add 2 mL of AML-12 medium to each well of a 6-well plate, then add 100 μL of cell suspension, gently shake to mix, label the plate information, and incubate at 37°C in a 5% CO2 incubator. Observe cell adhesion and distribution under a microscope after 24 hours.

[0039] The above AML-12 culture medium formula is as follows: 500 mL F12 medium, 500 μL dexamethasone, 5 mL insulin, 50 mL FBS (fetal bovine serum), and 5 mL of penicillin + streptomycin.

[0040] (2) Cell-based drug delivery

[0041] After the cells have grown to 60-70% confluence, gently wash twice with PBS buffer preheated in a 37°C water bath to remove the old culture medium.

[0042] The drug delivery system was established using six-well plates. Each group was given 3 mL of drug delivery medium (comprising 90% AML-12 medium + 10% F12 medium). The control group was given 3 μL each of 20% BSA and 40% BSA, while the model group was given 3 μL each of OA-d-BSA and PA-d-BSA. The drug administration groups are based on the modeling groups with different concentrations of drugs added. For example: blank group = 3 mL drug substrate + 3 μL 20% BSA + 3 μL 40% BSA; modeling group = 3 mL drug substrate + 3 μL OA-d-BSA + 3 μL PA-d-BSA; 2.5 μM salvianolic acid B drug administration group = 3 mL drug substrate + 3 μL OA-d-BSA + 3 μL PA-d-BSA + 3 μL 2.5 mM salvianolic acid B (the final concentration of salvianolic acid B in 3 mL drug substrate is 2.5 μM).

[0043] The specific dosages for each administration group are shown in Table 1. After administration, the culture plates were placed in a 37℃, 5% CO2 incubator for 24 h.

[0044] Table 1. Dosage of hyperoside, salvianolic acid B, and combined drug administration groups in each group

[0045]

[0046] 3. Triglycerides (TG)

[0047] Remove the 6-well cell culture plate from the incubator, aspirate the culture medium, add 1 mL of PBS buffer to each well and wash twice. After washing, add 200 μL of PBS buffer to each well, then scrape the cells off with a cell scraper and sonicate them under ice-water bath conditions (25%, 5 seconds, 30-second interval, 3 cycles). The prepared homogenate is measured directly without centrifugation.

[0048] Protein content was determined using the BCA method, and TG content was determined according to the experimental procedures provided by Nanjing Jiancheng Biotechnology Reagent Kit.

[0049] The results are as follows Figure 1 As shown, the experimental results indicate that both hyperoside and salvianolic acid B groups have a certain alleviating effect on OA- and PA-induced lipid accumulation at the cellular level. When hyperoside and salvianolic acid B are administered in combination, the Sal B 2.5μM+Hyp 3.75μM, Sal B 5μM+Hyp 7.5μM, and Sal B 10μM+Hyp 15μM groups showed better effects compared to any one of the single-drug groups, suggesting a possible synergistic effect. The synergistic effect will be further analyzed using the Chou-Talalay combination index.

[0050] Example 2

[0051] Chou-Talalay combination index analysis of drug combination therapy

[0052] The Chou-Talalay method is based on the median-effect principle, which assumes that the dose-response curve of a drug follows a specific mathematical model. When two drugs are used in combination, the actual effect of the combined medication is compared with the theoretical additive effect to determine whether the drugs have a synergistic, additive, or antagonistic effect. It is a commonly used method for assessing the interactions of two or more drugs used in combination.

[0053] Specifically, when CI < 1, it indicates that the effect of combined drug use is greater than the simple sum of the effects of the two drugs used alone, that is, there is a synergistic effect between the drugs; when CI = 1, it indicates that the effect of combined drug use is equal to the sum of the effects of the two drugs used alone, that is, an additive effect; when CI > 1, it indicates that the effect of combined drug use is less than the sum of the effects of the two drugs used alone, that is, there is an antagonistic effect between the drugs.

[0054] Analysis Procedure: Using the model group as the standard, the ratio of the drug group to the model group is taken as the proportion of the drug group. 1 minus this value is taken as the effect value (Fa value, or inhibition rate) of the drug group. The calculation of the CI value depends on the drug's effect value (inhibition rate). The corresponding effect values ​​for each group are shown in Table 2. When the effect value is negative, it is recorded as 0 when calculating the CI value. The effect curves of the two single drugs are calculated using CompuSyn software, and finally fitted to the curve of the combination group. The calculation formula is as follows: ,in, fa Indicates the effect size (inhibition rate). fu Indicates the unaffected rate. D This indicates the dosage of a drug when used in combination therapy. Dm This represents the equivalent dose when used alone, calculated as follows. m This is the CI value; the calculation results are shown in Table 3.

[0055] Table 2. Dosage and effect values ​​for each group

[0056]

[0057] Table 3. Fa and CI values ​​of the combined group

[0058]

[0059] The CI values ​​for combined drug therapy are shown in Table 3. The results generated by CompuSyn software are as follows: Figure 2 As shown, the Chou-Talalay combination index analysis revealed that this synergistic effect is significantly concentration-dependent. In the low-concentration combination groups (combination groups 1 and 2), the CI value was less than 1, indicating that the two drugs exhibited a synergistic effect, meaning that the combined effect was superior to the sum of the effects of the individual drugs, and could more effectively inhibit TG levels. However, no synergistic effect was observed in the high-concentration combination groups (combination groups 3-5). The CI values ​​confirm that hyperoside and salvianolic acid B in the low-concentration region can synergistically reduce lipid accumulation levels at the cellular level, providing important experimental evidence for subsequent studies.

[0060] Example 3

[0061] Animal-level experiments

[0062] Male C57BL / 6 mice (purchased from Xingkang Biotechnology), SPF grade, weighing 20±2g, were randomly divided into 5 groups (n=8 per group) after one week of acclimatization: Control group, Model group, Sal B (10mg / kg), Hyperoside (1.5mg / kg), and Combined treatment group (Sal B+Hyp, Sal B 10mg / kg + Hyp 1.5mg / kg). Except for the Control group, which was fed a standard diet, the other groups were fed a Western diet for 16 weeks to induce a non-alcoholic fatty liver disease model. During the experiment, while establishing the model, each group of mice was administered either a blank solution or the corresponding drug solution by gavage, and the weight data of each group of mice were recorded weekly. After 16 weeks of modeling and drug administration, liver tissue was collected for testing. The mice were fasted for 12 hours the day before the experiment, and blood was collected through the orbital venous plexus. After blood collection, the mice were euthanized, and liver tissue was collected.

[0063] 1. Effects of each drug administration group on TG content in mouse liver

[0064] Sample processing was strictly performed according to the instructions of the Nanjing Jiancheng TG reagent kit. The liver was weighed and anhydrous ethanol was added at a ratio of 1:9 (g / mL). Then, steel balls were added to grind the liver. After grinding, the steel balls were removed and the sample was centrifuged at 12,000 rpm for 10 min. The supernatant was then obtained for detection.

[0065] The results of TG content detection in the livers of mice in each group are as follows: Figure 3 As shown, compared with the control group, the liver TG content in the model group was significantly increased, indicating successful modeling. The TG content in the salvianolic acid B administration group was higher than that in the model group, indicating that this concentration would aggravate fatty liver in mice. The liver TG content in the hyperoside administration group was lower than that in the model group. In the combined administration group of hyperoside and salvianolic acid B, the liver TG content decreased significantly, indicating that there was a synergistic effect of combined administration.

[0066] 2. Liver H&E staining

[0067] Paraffin embedding and sectioning: After fixation with 4% paraformaldehyde for 24 hours, mouse liver tissue was removed and placed in a tissue embedding cassette. The tissue was then dehydrated using an automated dehydrator. After dehydration, the tissue was transferred to a paraffin embedding machine for embedding, with the liver tissue positioned centrally and encapsulated in paraffin as much as possible. After the paraffin solidified, the tissue was stored at room temperature. For sectioning, the paraffin block was fixed on a pathological microtome, and the section thickness was set to 4 μm. After sectioning, the paraffin section was carefully lifted with a glass slide, laid flat on a slide box, and then baked in a 70℃ oven for 30 minutes. After natural cooling, the section was stored at room temperature. Dewaxing to water: The paraffin sections were baked in a 70℃ oven for 1 hour, and then immediately placed in a paraffin embedding machine. Immerse the sections in xylene I for 10 minutes, then in 100%, 95%, and 85% ethanol for 10 minutes each, and wash three times with distilled water. For staining, immerse the sections in hematoxylin solution for 5 minutes, wash three times with distilled water, then differentiate with 1% hydrochloric acid alcohol for 3 seconds, and wash three times with distilled water. Place them in lithium carbonate blue solution for 3 seconds, then rinse with distilled water for 15 minutes. Finally, stain with eosin solution for 3 minutes and rinse with distilled water. For dehydration and mounting, immerse the sections in 75%, 85%, 95%, and 100% ethanol for 5 minutes each, then in xylene I and xylene II for 5 minutes each. After air-drying, mount with neutral resin. Observe and photograph under a regular optical microscope.

[0068] The results are as follows Figure 4 As shown, the liver cells in the control group had normal morphology and were neatly arranged, while the liver cells in the model group had disordered structure and obvious pathological changes of fatty degeneration. The Danshensu B group showed a tendency to aggravate pathological damage, while the pathological damage of liver cells in the hyperoside group was improved to a certain extent compared with the model group, but some abnormalities still existed. The liver cells in the combined drug group had morphology and arrangement closer to the control group, and the improvement of pathological damage was more significant.

[0069] 3. Liver oil red O assay

[0070] Fixation of fresh frozen sections: The frozen sections were thawed and fixed in fixative for 15 minutes, rinsed with tap water and air-dried; Oil red staining: The sections were immersed in oil red stain for 8 minutes, and the sections were covered to avoid light during this process; Background differentiation: The sections were removed from the staining tank, held in the air for 3 seconds and then immersed in 60% isopropanol twice for differentiation, for 3 seconds and 5 seconds respectively. Next, immerse the slides twice in pure water for 10 seconds each time; for hematoxylin staining: after rinsing in pure water, remove the slides, let them air dry for 3 seconds, then immerse them in hematoxylin for counterstaining for 5 minutes, then immerse them three times in pure water for 5 seconds, 10 seconds, and 30 seconds respectively; differentiate in differentiation solution (using 60% alcohol as solvent) for 5 seconds, then immerse them twice in distilled water for 10 seconds each time, and then in blue solution for 1 second. Gently immerse the slides twice in tap water for 5 seconds and 10 seconds respectively, and examine the staining effect under a microscope; mounting: mount the slides with glycerol gelatin mounting medium; image acquisition: lipid droplets appear bright red, and cell nuclei appear purple-blue.

[0071] The results are as follows Figure 5As shown, compared with the model group, the red lipid droplet content in the salvianolic acid B group was increased, while the red lipid droplet content in the hyperoside group was decreased. The combination of salvianolic acid B and hyperoside can significantly reduce the lipid content in the liver, and has a significant improvement effect compared with the administration of salvianolic acid B or hyperoside alone, indicating that the combination of salvianolic acid B and hyperoside can synergistically improve lipid accumulation in NAFLD.

[0072] 3. Serum AST activity detection

[0073] Serum AST (aspartate aminotransferase) activity is an important indicator of the degree of liver damage. When hepatocytes are damaged, AST is released into the bloodstream, leading to an increase in serum AST activity. Serum AST activity was measured using a reagent kit from Nanjing Jiancheng Biotechnology Co., Ltd.

[0074] The results are as follows Figure 6 As shown, compared with the control group, the serum AST activity of mice in the model group was increased, indicating that the liver injury model was successfully established. The AST activity in the salvianolic acid B group was further increased, indicating that the administration of salvianolic acid B alone at this concentration would aggravate liver injury. The AST activity in the hyperoside group and the combined administration group was decreased. Among them, the combined administration can significantly reduce the hepatic steatosis and inflammatory pathological response in NAFLD mice, enhance anti-inflammatory activity, and synergistically improve liver injury.

[0075] In this application, the inventors discovered during their research that hyperoside and salvianolic acid B can synergistically reduce TG levels at the cellular level. In animal experiments, when the two drugs were used alone for NAFLD, their effects on different indicators varied significantly, making it difficult to achieve a comprehensive and stable therapeutic effect. Specifically, while hyperoside could reduce liver TG levels and serum AST activity, the effect was not significant. While salvianolic acid B showed some mitigation of lipid accumulation in cellular experiments, in mouse experiments, administration alone at specific concentrations led to increased liver TG levels and exacerbated liver tissue pathological damage. This indicates that hyperoside or salvianolic acid B, when used alone, cannot simultaneously achieve a balance between liver protection and systemic lipid metabolism. This study, through the combined use of the drugs, overcomes the side effects or adverse effects of using either drug alone, significantly reducing liver TG accumulation and inflammatory responses. Further analysis of CI values ​​from cellular experiments confirmed that the two drugs have a synergistic effect in reducing liver lipids and inflammation.

Claims

1. The application of a drug for the treatment and / or prevention of NAFLD prepared by combining hyperoside and salvianolic acid B, characterized in that, The concentration of salvianolic acid B is 2.5~5μM, the concentration of hyperoside is 3.75~7.5μM, and the molar ratio of salvianolic acid B to hyperoside is 2:

3.

2. A medicament for the prevention and / or treatment of NAFLD, characterized in that, The active ingredients of the drug include hyperoside and salvianolic acid B, wherein the concentration of salvianolic acid B is 2.5~5μM, the concentration of hyperoside is 3.75~7.5μM, and the molar ratio of salvianolic acid B to hyperoside is 2:

3.

3. The medicament for the prevention and / or treatment of NAFLD as described in claim 2, characterized in that, The drug contains one or more pharmaceutically acceptable carriers or excipients.

4. The medicament for the prevention and / or treatment of NAFLD as described in claim 2, characterized in that, The drug is available in oral liquid or injection form.

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