A ginsenoside Rg5 derivative, its synthesis method and application

Abstract

This invention discloses a ginsenoside Rg5 derivative with the molecular formula: C 42 H 74 O 12 The molecular weight is 770.5180. Furthermore, this invention also discloses the synthesis method and application of this derivative. The synthesis method of this invention is simple and safe, and the synthesized ginsenoside Rg5 derivative exhibits strong stability and superior therapeutic effect on non-alcoholic steatohepatitis (NAH). This invention is the first to combine the prepared ginsenoside Rg5 derivative with the PDE4 inhibitor (R)-(-)-Rolipram, showing significantly better efficacy than either drug alone. When the molar ratio of the two drugs is 1:1, the drug exhibits even better therapeutic effect on NHA, showing promising application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a ginsenoside Rg5 derivative, its synthesis method, and its application. Background Technology

[0002] Nonalcoholic steatohepatitis (NAHH) is a chronic metabolic disease with a complex pathogenesis and an increasing prevalence, posing a significant burden on global health. NHAH is mainly characterized by lipid metabolism disorders, hepatic lipid accumulation, hepatic oxidative stress, and liver fibrosis, among other things.

[0003] Ginsenoside Rg5 exhibits good pharmacological activity in treating non-alcoholic steatohepatitis (NAH). However, ginsenoside Rg5 has poor stability under normal pressure and heating conditions. Its stability is affected by factors such as light, heat, and oxygen, making it difficult to preserve and posing a major challenge in research.

[0004] Therefore, providing a drug that is highly stable and can effectively treat non-alcoholic steatohepatitis is an urgent problem to be solved. Summary of the Invention

[0005] The technical problem to be solved by this invention is to address the shortcomings of the prior art by providing a ginsenoside Rg5 derivative. This derivative exhibits strong stability and superior therapeutic effects against non-alcoholic steatohepatitis (NAH). When combined with the PDE4 inhibitor (R)-(-)-Rolipram, it demonstrates even better therapeutic efficacy against NHA and shows promising application prospects.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a ginsenoside Rg5 derivative, characterized in that it has the following structural formula:

[0007]

[0008] The molecular formula is: C 42 H 74 O 12 The molecular weight is 770.5180.

[0009] Furthermore, the present invention provides a method for synthesizing the above-mentioned ginsenoside Rg5 derivative, characterized by comprising the following steps:

[0010] Step 1: Dissolve ginsenoside Rg5 in anhydrous ethanol, then add palladium catalyst on carbon, followed by an aqueous solution of sodium hypophosphite monohydrate. React at room temperature. After the reaction is complete, filter to remove the palladium catalyst on carbon, extract with dichloromethane, wash the organic phase with saturated brine, dry with anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain the crude product.

[0011] Step 2: Dissolve the crude product from Step 1 in anhydrous ethanol, purify it by semi-preparative liquid chromatography, collect the eluent of ginsenoside Rg5 derivative, and dry the collected eluent by rotary evaporation to obtain the product ginsenoside Rg5 derivative.

[0012] The above method is characterized in that, in step one, the mass ratio of ginsenoside Rg5 to the volume of anhydrous ethanol is 1:(4-8), where the mass unit is g and the volume unit is mL; the mass of the palladium on carbon catalyst is 3%-5% of the mass of ginsenoside Rg5, and the mass of sodium hypophosphite monohydrate is 40%-80% of the mass of ginsenoside Rg5.

[0013] The method described above is characterized in that the reaction time in step one is 15h to 30h.

[0014] The method described above is characterized in that the separation conditions for the semi-preparative liquid chromatography in step two are as follows: C18 column (20×250mm, 10μm), UV detector, detection wavelength of 203nm, flow rate of 10mL / min, mobile phase A is pure water, mobile phase B is acetonitrile, and gradient elution program is 0~100min, 45%~55% acetonitrile.

[0015] Furthermore, the present invention provides an application of the above-mentioned ginsenoside Rg5 derivative in the preparation of a drug for treating non-alcoholic steatohepatitis.

[0016] The above-described application is characterized in that the drug includes a ginsenoside Rg5 derivative.

[0017] The above-described application is characterized in that the drug further includes a PDE4 inhibitor.

[0018] The above application is characterized in that the PDE4 inhibitor is (R)-(-)-Rolipram.

[0019] The above application is characterized in that the molar ratio of ginsenoside Rg5 derivative to (R)-(-)-Rolipram in the drug is 1:1.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] 1. The synthesis method of the present invention is simple and safe, and the synthesized ginsenoside Rg5 derivative has strong stability and better therapeutic effect on non-alcoholic steatohepatitis than ginsenoside Rg5.

[0022] 2. This invention is the first to combine the prepared ginsenoside Rg5 derivative with the PDE4 inhibitor (R)-(-)-Rolipram, and the effect is significantly better than that of the single drug. When the molar ratio of the two is 1:1, the drug has a better effect in treating non-alcoholic steatohepatitis and has good application prospects.

[0023] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the synthetic route for the ginsenoside Rg5 derivative of the present invention.

[0025] Figure 2 The ginsenoside Rg5 derivative of the present invention 1 H-NMR spectrum.

[0026] Figure 3 The ginsenoside Rg5 derivative of the present invention 13 C-NMR spectrum.

[0027] Figure 4 This is a high-resolution mass spectrum of the ginsenoside Rg5 derivative of the present invention.

[0028] Figure 5 The figure shows the results of the stability test of the ginsenoside Rg5 derivative and ginsenoside Rg5 of the present invention.

[0029] Figure 6 The effect of different ginsenoside treatments on the proliferation activity of FFA-HepG2 cells (*p<0.05, **p<0.01, ***p<0.005).

[0030] Figure 7 The effect of different ginsenoside treatments on TG content in FFA-HepG2 cells (*p<0.05, **p<0.01, ***p<0.005).

[0031] Figure 8 Figure showing the effect of different ginsenoside treatments on improving lipid accumulation in FFA-HepG2 cells.

[0032] Figure 9 The figure shows the effect of different treatment groups on the proliferation activity of FFA-HepG2 cells.

[0033] Figure 10 The effect of different treatment groups on TG content in FFA-HepG2 cells and the synergistic CI value.

[0034] Figure 11The effect of different treatment groups on the content of inflammatory factors secreted by FFA-HepG2 cells (*p<0.05, **p<0.01, ***p<0.005).

[0035] Figure 12 Figure 1 shows the effect of different treatment groups on improving lipid accumulation in FFA-HepG2 cells.

[0036] Figure 13 Images showing the results of Oil Red O staining of liver tissue from different treatment groups.

[0037] Figure 14 Images showing the H&E staining results of liver tissue from different treatment groups.

[0038] Figure 15 Images showing Masson staining results of liver tissue from different treatment groups.

[0039] Figure 16 The effect of different treatment groups on serum CHO and TG levels in mice (*p<0.05, **p<0.01, ***p<0.005). Detailed Implementation

[0040] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention are described in detail below with reference to examples. In the following examples, ginsenoside Rg5 (purity >97%) was provided by the Institute of Biological and Pharmaceutical Sciences, Northwest University; palladium on carbon catalyst, part number P821216, was purchased from Maclean's (Shanghai, China); and a Waters C18 column (20×250mm, 10μm) was used. Unless otherwise specified, all reagents used in the description of the present invention are commercially available.

[0041] The ginsenoside Rg5 derivative of the present invention has the following structural formula:

[0042]

[0043] The molecular formula is: C 42 H 74 O 12 The molecular weight is 770.5180.

[0044] See the synthetic route. Figure 1 The synthesis method includes the following steps:

[0045] Step 1: Dissolve ginsenoside Rg5 in anhydrous ethanol, then add palladium catalyst on carbon, followed by slow addition of an aqueous solution of sodium hypophosphite monohydrate. React at room temperature. After the reaction is complete, filter to remove the palladium catalyst on carbon, extract with dichloromethane, wash the organic phase with saturated brine, dry with anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain the crude product. The mass ratio of ginsenoside Rg5 to the volume of anhydrous ethanol is 1:(4-8), where mass is in g and volume is in mL. The mass of palladium catalyst on carbon is 3%-5% of the mass of ginsenoside Rg5, and the mass of sodium hypophosphite monohydrate is 40%-80% of the mass of ginsenoside Rg5. The reaction time is 15-30 h.

[0046] Step 2: Dissolve the crude product from Step 1 in anhydrous ethanol and purify it using semi-preparative liquid chromatography. Collect the eluent of ginsenoside Rg5 derivative, and dry the collected eluent by rotary evaporation to obtain the product ginsenoside Rg5 derivative. The separation conditions for semi-preparative liquid chromatography are: C18 column (20×250mm, 10μm), UV detector, detection wavelength of 203nm, flow rate of 10mL / min, mobile phase A is pure water, mobile phase B is acetonitrile, and gradient elution program is 0-100min, 45%-55% acetonitrile.

[0047] Example 1

[0048] The specific synthesis method of the ginsenoside Rg5 derivative in this embodiment includes:

[0049] Step 1: Dissolve 500 mg of ginsenoside Rg5 in 3 mL of anhydrous ethanol, then add 20 mg of palladium catalyst on carbon, and slowly add an aqueous solution containing 300 mg of sodium hypophosphite monohydrate. React at room temperature for 20 h. After the reaction is complete, filter to remove the palladium catalyst on carbon, extract with dichloromethane, wash the organic phase with saturated brine, dry with anhydrous sodium sulfate, filter and concentrate under reduced pressure to obtain the crude product.

[0050] Step 2: Dissolve the crude product from Step 1 in anhydrous ethanol and purify it using semi-preparative liquid chromatography. Collect the eluent of ginsenoside Rg5 derivative, and dry the collected eluent by rotary evaporation to obtain the product ginsenoside Rg5 derivative. The separation conditions for semi-preparative liquid chromatography are: C18 column (20×250mm, 10μm), UV detector, detection wavelength of 203nm, flow rate of 10mL / min, mobile phase A is pure water, mobile phase B is acetonitrile, and gradient elution program is 0-100min, 45%-55% acetonitrile.

[0051] Example 2

[0052] The specific synthesis method of the ginsenoside Rg5 derivative in this embodiment includes:

[0053] Step 1: Dissolve 500 mg of ginsenoside Rg5 in 2 mL of anhydrous ethanol, then add 15 mg of palladium catalyst on carbon, and slowly add an aqueous solution containing 200 mg of sodium hypophosphite monohydrate. React at room temperature for 15 h. After the reaction is complete, filter to remove the palladium catalyst on carbon, extract with dichloromethane, wash the organic phase with saturated brine, dry with anhydrous sodium sulfate, filter and concentrate under reduced pressure to obtain the crude product.

[0054] Step 2: Dissolve the crude product from Step 1 in anhydrous ethanol and purify it using semi-preparative liquid chromatography. Collect the eluent of ginsenoside Rg5 derivative, and dry the collected eluent by rotary evaporation to obtain the product ginsenoside Rg5 derivative. The separation conditions for semi-preparative liquid chromatography are: C18 column (20×250mm, 10μm), UV detector, detection wavelength of 203nm, flow rate of 10mL / min, mobile phase A is pure water, mobile phase B is acetonitrile, and gradient elution program is 0-100min, 45%-55% acetonitrile.

[0055] Example 3

[0056] The specific synthesis method of the ginsenoside Rg5 derivative in this embodiment includes:

[0057] Step 1: Dissolve 500 mg of ginsenoside Rg5 in 4 mL of anhydrous ethanol, then add 25 mg of palladium catalyst on carbon, and slowly add an aqueous solution containing 400 mg of sodium hypophosphite monohydrate. React at room temperature for 30 h. After the reaction is complete, filter to remove the palladium catalyst on carbon, extract with dichloromethane, wash the organic phase with saturated brine, dry with anhydrous sodium sulfate, filter and concentrate under reduced pressure to obtain the crude product.

[0058] Step 2: Dissolve the crude product from Step 1 in anhydrous ethanol and purify it using semi-preparative liquid chromatography. Collect the eluent of ginsenoside Rg5 derivative, and dry the collected eluent by rotary evaporation to obtain the product ginsenoside Rg5 derivative. The separation conditions for semi-preparative liquid chromatography are: C18 column (20×250mm, 10μm), UV detector, detection wavelength of 203nm, flow rate of 10mL / min, mobile phase A is pure water, mobile phase B is acetonitrile, and gradient elution program is 0-100min, 45%-55% acetonitrile.

[0059] The proton and carbon spectra of ginsenoside Rg5 synthesized in Examples 1 to 3 of this invention are as follows: Figure 2 and Figure 3 As shown, the high-resolution mass spectrum is as follows: Figure 4 As shown, the data is as follows: white solid; 1H NMR (400MHz, MeOD) δ4.67(d,J=7.6Hz,1H),4.44(d,J=6.6Hz,1H),3.84(t,J=12.5Hz,2H),3.61(m,6H),3.36(m,2H),3.23 (m,6H),1.99(m,4H),1.83–1.36(m,14H),1.37–1.25(m,5H),1.18(m,5H),1.05(m,8H),0.98–0.83(m,17H),0.79(m,3H).

[0060] Example 4 Stability Test

[0061] 5 mg of dried ginsenoside Rg5 and the ginsenoside Rg5 derivative prepared in Example 1 of this invention (hereinafter referred to as ginsenoside Rg5a) were accurately weighed separately and diluted to 50 mL in a volumetric flask with chromatographically pure ethanol to obtain a standard solution. Several 1 mL HPLC vials were taken, and 1 mL of ginsenoside Rg5 and ginsenoside Rg5a standard solutions were added. Three parallel samples (a, b, and c) were prepared for each group and evaporated to dryness using a rotary evaporator at 30 °C to obtain the test sample. HPLC vials containing ginsenoside Rg5 and ginsenoside Rg5a standards were placed at 25 °C for 5, 10, 15, 20, and 25 days. The samples were dissolved in 1 mL of chromatographically pure ethanol, filtered through a 0.22 μm organic filter membrane, and analyzed by HPLC to investigate the changes in the stability of ginsenoside Rg5 and ginsenoside Rg5a.

[0062] The results are as follows Figure 5 As shown, after 25 days, the target peak area of ​​ginsenoside Rg5 decreased by approximately 90%, while the target peak area of ​​ginsenoside Rg5a decreased by less than 15%. This demonstrates that the stability of the derivatives was significantly improved.

[0063] Example 5

[0064] This embodiment analyzes the anti-inflammatory effects of ginsenoside Rg5a, ginsenoside Rg5, and ginsenoside Rh4 prepared in Example 1 through the following cell experiments.

[0065] 1. CCK8 cell proliferation assay to detect cell viability.

[0066] Take 1×10 4HepG2 cells in logarithmic growth phase were seeded in 96-well plates. The culture medium used was MEM cell culture medium containing 1% penicillin-streptomycin solution and 10% fetal bovine serum. The cells were incubated overnight at 37°C in a 5% CO2 incubator. After establishing a free fatty acid (FFA)-induced non-alcoholic fatty liver disease model in HepG2 cells according to known experimental conditions, the following groups were set up for treatment: model group, ginsenoside Rg5 group, ginsenoside Rg5a group, and ginsenoside Rh4 group.

[0067] Model group: An FFA-HepG2 cell model was established without drug treatment.

[0068] Ginsenoside Rg5 group / Ginsenoside Rg5a group / Ginsenoside Rh4 group: FFA-HepG2 cells were treated with different ginsenosides at final concentrations of 10μM, 20μM, 30μM, 40μM, and 50μM, respectively. The volume of drug added to each group was the same (1μL drug (or DMSO) / mL culture medium). After culturing the cells for 24 hours after drug addition, cell viability was detected using the CCK8 assay. The CCK8 stock solution was diluted 1:10 with serum-free MEM culture medium to prepare the working solution. The culture medium in the 96-well plate was aspirated, and 100μL of CCK8 working solution was added again. After incubation at 37℃ in the dark for 2 hours, the proliferation of FFA-HepG2 cells in each experimental group was detected and analyzed.

[0069] The results are as follows Figure 6 As shown, ginsenoside Rg5 and ginsenoside Rh4 groups, at concentrations of 40 μM or 50 μM, exhibited certain inhibitory effects on the proliferation of FFA-HepG2 cells; while ginsenoside Rg5a group showed no toxic side effects on FFA-HepG2 cells at the aforementioned concentrations. Therefore, ginsenoside Rg5a is less toxic than the other two. Subsequent studies will use a concentration of 50 μM to investigate the effects of different ginsenosides on FFA-HepG2 cells.

[0070] 2. Effects of different ginsenosides on triglyceride (TG) content in FFA-HepG2 cells

[0071] HepG2 cells in the logarithmic growth phase were seeded into 6-well plates to establish a free fatty acid (FFA)-induced non-alcoholic steatohepatitis (NASH) model. Then, blank control group, model group, ginsenoside Rg5 group, ginsenoside Rg5a group, and ginsenoside Rh4 group were set up for treatment.

[0072] Blank group: Add DMSO;

[0073] Model group: An FFA-HepG2 cell model was established without drug treatment.

[0074] Ginsenoside Rg5 group / Ginsenoside Rg5a group / Ginsenoside Rh4 group: FFA-HepG2 cells were treated with different ginsenosides at a final concentration of 50 μM;

[0075] The TG content in different experimental groups was detected using a kit. The results are as follows: Figure 7 As shown, ginsenoside Rg5a has the best effect in reducing FFA-induced intracellular TG content.

[0076] 3. Oil Red O staining

[0077] HepG2 cells in the logarithmic growth phase were seeded into 6-well plates to establish a free fatty acid (FFA)-induced non-alcoholic steatohepatitis (NASH) model. Then, blank control group, model group, ginsenoside Rg5 group, ginsenoside Rg5a group, and ginsenoside Rh4 group were set up for treatment.

[0078] Blank group: Add DMSO;

[0079] Model group: An FFA-HepG2 cell model was established without drug treatment.

[0080] Ginsenoside Rg5 group / Ginsenoside Rg5a group / Ginsenoside Rh4 group: FFA-HepG2 cells were treated with different ginsenosides at a final concentration of 50 μM;

[0081] Cells were stained using an Oil Red O staining kit to observe intracellular lipid accumulation.

[0082] The results are as follows Figure 8 As shown in the figure, FFA significantly induces lipid accumulation, manifested by the appearance of numerous lipid droplets in the model group cells, resulting in a significant increase in the red area compared to the control group. After drug treatment, the red area decreased, indicating a reduction in intracellular lipid accumulation. Among them, ginsenoside Rg5a showed the least red area after treatment, demonstrating its superior lipid-lowering effect.

[0083] Example 6

[0084] This embodiment analyzes the anti-inflammatory effects of (R)-(-)-Rolipram, ginsenoside Rg5a prepared in Example 1, and the combination of the two through the following cell experiments.

[0085] 1. CCK8 cell proliferation assay to detect cell viability.

[0086] Take 1×10 4HepG2 cells in logarithmic growth phase were seeded in 96-well plates. The culture medium used was MEM cell culture medium containing 1% penicillin-streptomycin solution and 10% fetal bovine serum. The cells were incubated overnight at 37°C in a 5% CO2 incubator. After establishing a free fatty acid (FFA)-induced non-alcoholic fatty liver disease model in HepG2 cells according to known experimental conditions, model groups, ginsenoside Rg5a groups, (R)-(-)-Rolipram groups, and ginsenoside Rg5a combined with (R)-(-)-Rolipram groups were set up, and different drugs were added for treatment.

[0087] Model group: An FFA-HepG2 cell model was established without drug treatment.

[0088] Ginsenoside Rg5a group: FFA-HepG2 cells were treated with ginsenoside Rg5a at final concentrations of 10μM, 20μM, 30μM, 40μM, and 50μM, respectively.

[0089] (R)-(-)-Rolipram group: FFA-HepG2 cells were treated with (R)-(-)-Rolipram at final concentrations of 10 μM, 20 μM, 30 μM, 40 μM, and 50 μM, respectively.

[0090] Ginsenoside Rg5a combined with (R)-(-)-Rolipram group: Ginsenoside Rg5a and (R)-(-)-Rolipram were added at a final concentration of 10 μM, or at a final concentration of 20 μM, or at a final concentration of 30 μM, or at a final concentration of 40 μM, or at a final concentration of 50 μM (the molar ratio of ginsenoside Rg5a to (R)-(-)-Rolipram was 1:1).

[0091] The volume of drug added to each group was the same (1 μL drug (or DMSO) / mL culture medium). After adding the drug and continuing to culture the cells for 24 hours, cell viability was detected using the CCK8 assay. The CCK8 stock solution was diluted 1:10 with serum-free MEM culture medium to prepare the working solution. After aspirating the culture medium from the 96-well plate, 100 μL of CCK8 working solution was added again, and the cells were incubated in a 37°C incubator in the dark for 2 hours. The proliferation of FFA-HepG2 cells in each experimental group was then analyzed.

[0092] The results are as follows Figure 9As shown, compared with the control group, ginsenoside Rg5a, (R)-(-)-Rolipram, and the combination of the two did not inhibit the proliferation of FFA-HepG2 cells, indicating that neither of the two compounds had any toxic side effects on FFA-HepG2 cells at the concentrations they were used at.

[0093] 2. Effects of different proportions of ginsenoside Rg5a combined with (R)-(-)-Rolipram on the triglyceride (TG) content in FFA-HepG2 cells, and synergistic effect analysis of the two.

[0094] HepG2 cells in the logarithmic growth phase were seeded in 6-well plates to establish a free fatty acid (FFA)-induced HepG2 non-alcoholic steatohepatitis model. Different drugs were added to treat the cells.

[0095] Blank group: Add DMSO;

[0096] Model group: An FFA-HepG2 cell model was established without drug treatment.

[0097] Ginsenoside Rg5a group: HepG2 cells were treated with ginsenoside Rg5a at final concentrations of 30 μM, 40 μM, 50 μM, and 60 μM, respectively.

[0098] (R)-(-)-Rolipram group: HepG2 cells were treated with (R)-(-)-Rolipram at a final concentration of 50 μM.

[0099] Ginsenoside Rg5a combined with (R)-(-)-Rolipram: Ginsenoside Rg5a was added at a final concentration of 30 μM and (R)-(-)-Rolipram at a final concentration of 50 μM, or ginsenoside Rg5a was added at a final concentration of 40 μM and (R)-(-)-Rolipram at a final concentration of 50 μM, or ginsenoside Rg5a was added at a final concentration of 50 μM and (R)-(-)-Rolipram at a final concentration of 60 μM and (R)-(-)-Rolipram at a final concentration of 50 μM. The CI values ​​for different combinations were calculated.

[0100] The results are as follows Figure 10 The synergistic effect was best when used in combination at a molar ratio of 1:1. Subsequent experiments used ginsenoside Rg5a at a final concentration of 50 μM and (R)-(-)-Rolipram at a final concentration of 50 μM.

[0101] 3. Oil Red O staining and inflammatory factor detection

[0102] HepG2 cells in the logarithmic growth phase were seeded in 6-well plates to establish a free fatty acid (FFA)-induced HepG2 non-alcoholic steatohepatitis model. Different drugs were added to treat the cells.

[0103] Blank group: Add DMSO;

[0104] Model group: An FFA-HepG2 cell model was established without drug treatment.

[0105] Ginsenoside Rg5a group: Cells were treated with ginsenoside Rg5a at a concentration of 50 μM;

[0106] (R)-(-)-Rolipram group: cells were treated with 50 μM (R)-(-)-Rolipram;

[0107] Ginsenoside Rg5a combined with (R)-(-)-Rolipram group: Cells were treated with 50 μM ginsenoside Rg5a + 50 μM (R)-(-)-Rolipram.

[0108] Subsequently, the intracellular lipid accumulation was detected using an Oil Red O staining kit; the levels of IL-6 and TNF-α in different experimental groups were detected using an ELISA kit.

[0109] The results are as follows Figure 11 , Figure 12 As shown, from Figure 11 It can be seen that after modeling, cells showed significant lipid accumulation, which decreased after drug treatment; in the combined drug group, lipid accumulation was significantly reduced. Figure 12 It can be seen that the secretion of IL-6 and TNF-α by cells in the model group was significantly increased compared with that in the blank control group. After intervention in the combined drug group, the secretion of the above inflammatory factors was significantly reduced.

[0110] Example 7 Animal Experiment

[0111] High-fat, high-sugar (HFHC) diet-induced non-alcoholic steatohepatitis (NAH) in mice: Six-week-old male C57BL / 6J mice were randomly divided into four groups: normal group, model group, ginsenoside Rg5a group, (R)-(-)-Rolipram group, and ginsenoside Rg5a combined with (R)-(-)-Rolipram group. The normal group was fed a standard diet. The other three groups were fed a high-fat diet (D12492) and given 4% sugar water (containing 45% sucrose and 55% fructose) for 8 consecutive weeks to establish a NHA model. Eight weeks later, the treatment groups were administered the drug via intraperitoneal injection for four weeks (ginsenoside Rg5a dose: 40 mg / kg, (R)-(-)-Rolipram group dose: 14 mg / kg, ginsenoside Rg5a combined with (R)-(-)-Rolipram group dose: ginsenoside Rg5a 20 mg / kg + (R)-(-)-Rolipram 7 mg / kg).

[0112] 1. Oil Red O staining of liver tissue:

[0113] After cryopreservation of liver tissue, the frozen tissue was excised and attached to a glass slide, then allowed to return to room temperature. After moistening with distilled water, it was rinsed with 60% isopropanol, followed by immersion in freshly prepared Oil Red O staining solution, and then rinsed thoroughly with distilled water. The slide was then mounted with water-soluble mounting medium and the junction between the slide and coverslip was sealed with neutral resin.

[0114] Oil Red O is a lipid-soluble dye that stains triglycerides and other neutral fats in tissues. Therefore, lipid droplets in tissue cells appear red, while cell nuclei appear blue. The percentage of red area in a section can reflect the severity of lipid accumulation. Results are as follows... Figure 13 As shown, the red area was the largest in the model group, while the red area was significantly reduced in the combination drug group, indicating a decrease in lipid accumulation.

[0115] 2. Liver tissue H&E staining:

[0116] After hydration, liver tissue sections were infiltrated with purified water, stained with hematoxylin, and rinsed with running water. The process involved differentiation, rinsing, hematoxylin staining, and rinsing again. After draining, the sections were stained with eosin for 30 seconds, rinsed, dried, mounted with neutral resin, examined under a microscope, and images were acquired for analysis.

[0117] H&E staining can reflect the degree of liver lesions, and the results are as follows: Figure 14 As shown, the model group exhibited central vein dilation, widened and disordered hepatic cords, partial sinusoidal congestion, and indistinct hepatocyte boundaries. After combined drug treatment, the liver lobule structure tended to return to normal, necrotic cells were significantly reduced, and fat vacuoles shrank, significantly improving the severity of liver lesions.

[0118] 3. Masson staining of liver tissue

[0119] After dewaxing paraffin sections to water, they were stained with hematoxylin for 5 minutes, then differentiated, rinsed with distilled water, and the blued areas were counterstained with Ponceau S, rinsed, and counterstained with aniline blue. Immediately afterwards, they were placed in 1% glacial acetic acid for dehydration, cleared, and then mounted with neutral resin.

[0120] Masson staining can be used to differentiate between collagen fibers and muscle fibers. In the staining results, collagen fibers appear blue, and muscle fibers appear red. The results are as follows... Figure 15 As shown, the normal group exhibited normal liver lobule structure, while the model group showed coarse, dark blue fibers in the portal area of ​​the liver according to Masson's trichrome staining, indicating liver fibrosis. Hepatocyte steatosis was also observed. After combined drug treatment, the area of ​​blue staining was significantly reduced, and the distribution was limited to the periphery of the blood vessel walls, indicating a significant improvement in the degree of liver fibrosis.

[0121] 4. Serum cholesterol (CHO) and triglyceride (TG) content in mice

[0122] To investigate the effects of drug action on lipid abnormalities induced by non-alcoholic steatohepatitis, the expression levels of cholesterol CHO and TG in mouse serum were assessed.

[0123] The level of triglycerides (TG) in the liver can directly reflect the lipid-lowering effect of drugs, as shown in the following results. Figure 16 As shown in the figure, ginsenoside Rg5a (a) and (b) represent (R)-(-)-Rolipram. The CHO and TG levels in the model group were significantly increased compared to the control group. Combined drug treatment could reduce the CHO and TG levels in mouse serum to normal levels.

[0124] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the present invention. Any simple modifications, alterations, or equivalent structural changes made to the above embodiments based on the technical essence of the invention shall still fall within the protection scope of the present invention.

Claims

1. Application of ginsenoside Rg5 derivative in the preparation of a drug for treating non-alcoholic steatohepatitis, characterized in that, The medicine comprises a ginsenoside Rg5 derivative and a PDE4 inhibitor (R)-(-)-Rolipram; the ginsenoside Rg5 derivative has a molecular formula of C 42 H 74 O 12 ; a molecular weight of 770.5180; and a structural formula as shown in the following formula (I): 。 2.The use of ginsenoside Rg5 derivative according to claim 1 in the preparation of a medicament for treating non-alcoholic steatohepatitis, characterized in that, The molar ratio of the ginsenoside Rg5 derivative to (R)-(-)-Rolipram in the medicament is 1:

1. The molar ratio of the ginsenoside Rg5 derivative to (R)-(-)-Rolipram in the medicament is 1:1.

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