Ginsenoside Rg5 derivative as well as synthesis method and application thereof

Abstract

The invention discloses a ginsenoside Rg5 derivative. The molecular formula of the ginsenoside Rg5 derivative is C42H74O12. And the molecular weight of the polymer is 770.5180. In addition, the invention also discloses a synthesis method and application of the derivative. The synthesis method is simple and safe, the synthesized ginsenoside Rg5 derivative is high in stability, and the treatment effect of the ginsenoside Rg5 derivative on non-alcoholic steatohepatitis is better than that of ginsenoside Rg5. The prepared ginsenoside Rg5 derivative and the PDE4 inhibitor (R)-(-)-Rolipram are compounded for the first time, the effect is remarkably better than that of a single medicine, and when the mole ratio of the ginsenoside Rg5 derivative to the PDE4 inhibitor (R)-(-)-Rolipram is 1: 1, the medicine has a better effect of treating non-alcoholic steatohepatitis and has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a ginsenoside Rg5 derivative and a synthesis method and application thereof. Background Art

[0002] Nonalcoholic fatty liver disease is a chronic metabolic disease with a complex pathogenesis and an increasing prevalence, which has caused a significant burden on global health. Nonalcoholic fatty liver disease is mainly manifested by lipid metabolism disorders, liver lipid accumulation, liver oxidative stress, liver fibrosis, etc.

[0003] Ginsenoside Rg5 has good pharmacological activity in treating non-alcoholic fatty liver disease. However, ginsenoside Rg5 has poor stability under normal pressure and heat conditions. Its stability is reduced by factors such as light, heat, and oxygen, and it is difficult to preserve, which is a major problem in the research process.

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

[0005] The technical problem to be solved by the present invention is to provide a ginsenoside Rg5 derivative in view of the above-mentioned deficiencies of the prior art. The derivative has strong stability, is better than ginsenoside Rg5 in the treatment of non-alcoholic fatty liver disease, and is compounded with the PDE4 inhibitor (R)-(-)-Rolipram to have a better effect in the treatment of non-alcoholic fatty liver disease, and has a good application prospect.

[0006] In order to solve the above 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 ; Molecular weight: 770.5180.

[0009] Furthermore, the present invention provides a method for synthesizing the above-mentioned ginsenoside Rg5 derivative, characterized in that it comprises the following steps:

[0010] Step 1, dissolving ginsenoside Rg5 in anhydrous ethanol, then adding palladium carbon catalyst, and then adding an aqueous solution of sodium hypophosphite monohydrate, reacting at room temperature, filtering to remove the palladium carbon catalyst after the reaction is completed, extracting with dichloromethane, washing the organic phase with saturated brine, drying with anhydrous sodium sulfate, filtering and concentrating under reduced pressure to obtain a crude product;

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

[0012] The above method is characterized in that the ratio of the mass of ginsenoside Rg5 to the volume of anhydrous ethanol in step 1 is 1:(4-8), wherein the unit of mass is g and the unit of volume is mL; the mass of the palladium-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 above method is characterized in that the reaction time in step 1 is 15h to 30h.

[0014] The above method is characterized in that the separation conditions of the semi-preparative liquid chromatography in step 2 are: C18 column (20×250 mm, 10 μm), UV detector, detection wavelength of 203 nm, volume flow rate of 10 mL / min, mobile phase A is pure water, B is acetonitrile, and gradient elution program: 0 to 100 min, 45% to 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 fatty liver disease.

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

[0017] The above application is characterized in that the drug also 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. The synthesized ginsenoside Rg5 derivative has strong stability and is better than ginsenoside Rg5 in the treatment of non-alcoholic fatty liver disease.

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

[0023] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The schematic diagram is a synthetic route of 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 It is a high-resolution mass spectrum of the ginsenoside Rg5 derivative of the present invention.

[0028] Figure 5 This is a graph showing the stability investigation results of the ginsenoside Rg5 derivatives and ginsenoside Rg5 of the present invention.

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

[0030] Figure 7 This is the effect of different ginsenoside treatment groups on the TG content of FFA-HepG2 cells (*p<0.05, **p<0.01, ***p<0.005).

[0031] Figure 8 This is a diagram showing the improvement of lipid accumulation in FFA-HepG2 cells by different ginsenoside treatment groups.

[0032] Fig. 9 This is a graph showing the effects of different treatment groups on the proliferation activity of FFA-HepG2 cells.

[0033] Fig.10 This is the effect diagram of different treatment groups on the TG content of FFA-HepG2 cells and the synergistic CI value.

[0034] Fig.11This is a graph showing the effects of different treatment groups on the levels of inflammatory factors secreted by FFA-HepG2 cells (*p<0.05, **p<0.01, ***p<0.005).

[0035] Fig.12 This is a diagram showing how different treatment groups improve lipid accumulation in FFA-HepG2 cells.

[0036] Fig.13 Oil red O staining results of liver tissue in different treatment groups.

[0037] Fig.14 The results of H&E staining of liver tissues in different treatment groups.

[0038] Fig.15 The results of Masson staining of liver tissue in different treatment groups.

[0039] Fig.16 The figure shows the effect of different treatment groups on the CHO and TG levels in mouse serum (*p<0.05, **p<0.01, ***p<0.005). DETAILED DESCRIPTION

[0040] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the examples. In the following examples, ginsenoside Rg5 (purity> 97%) was provided by the Institute of Biology and Medicine of Northwest University; palladium carbon catalyst, No. P821216, purchased from McLean (Shanghai, China); Waters C18 column (20×250mm, 10μm). In the description of the present invention, unless otherwise specified, the reagents used are all 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 ; Molecular weight: 770.5180.

[0044] Synthesis route see Figure 1 , the synthesis method comprises the following steps:

[0045] Step 1, dissolving ginsenoside Rg5 in anhydrous ethanol, then adding palladium carbon catalyst, and then slowly adding an aqueous solution of sodium hypophosphite monohydrate, reacting at room temperature, filtering to remove the palladium carbon catalyst after the reaction is completed, extracting with dichloromethane, washing the organic phase with saturated brine, drying with anhydrous sodium sulfate, filtering and concentrating under reduced pressure to obtain a crude product; the ratio of the mass of ginsenoside Rg5 to the volume of anhydrous ethanol is 1: (4-8), wherein the unit of mass is g, and the unit of volume is mL; the mass of the palladium carbon catalyst is 3%-5% of the mass of ginsenoside Rg5, and the mass of the sodium hypophosphite monohydrate is 40%-80% of the mass of ginsenoside Rg5; the reaction time is 15h-30h;

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

[0047] Example 1

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

[0049] Step 1, dissolve 500 mg of ginsenoside Rg5 in 3 mL of anhydrous ethanol, then add 20 mg of palladium carbon catalyst, and then slowly add an aqueous solution containing 300 mg of sodium hypophosphite monohydrate, react at room temperature for 20 hours, filter to remove the palladium carbon catalyst after the reaction, extract with dichloromethane, wash the organic phase with saturated brine, dry with anhydrous sodium sulfate, filter and concentrate under reduced pressure to obtain a crude product;

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

[0051] Example 2

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

[0053] Step 1, dissolve 500 mg of ginsenoside Rg5 in 2 mL of anhydrous ethanol, then add 15 mg of palladium carbon catalyst, and then slowly add an aqueous solution containing 200 mg of sodium hypophosphite monohydrate, react at room temperature for 15 hours, filter to remove the palladium carbon catalyst after the reaction, extract with dichloromethane, wash the organic phase with saturated brine, dry with anhydrous sodium sulfate, filter and concentrate under reduced pressure to obtain a crude product;

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

[0055] Example 3

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

[0057] Step 1, dissolve 500 mg of ginsenoside Rg5 in 4 mL of anhydrous ethanol, then add 25 mg of palladium carbon catalyst, and then slowly add an aqueous solution containing 400 mg of sodium hypophosphite monohydrate, react at room temperature for 30 hours, filter to remove the palladium carbon catalyst after the reaction, extract with dichloromethane, wash the organic phase with saturated brine, dry with anhydrous sodium sulfate, filter and concentrate under reduced pressure to obtain a crude product;

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

[0059] The hydrogen and carbon spectra of the compound ginsenoside Rg5 synthesized in Examples 1 to 3 of the present invention are as follows: Figure 2 and Figure 3 As shown in the high-resolution mass spectrum Figure 4 As shown, the data are 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 the present invention (hereinafter referred to as ginsenoside Rg5a) were accurately weighed, and the 50 mL volumetric flask was fixed to volume with chromatographic pure ethanol to obtain a standard solution. Take several 1 mL liquid phase vials, add 1 mL of ginsenoside Rg5 and ginsenoside Rg5a standard solutions, three parallel samples a, b, c in each group, and evaporate to dryness with a rotary evaporator at 30 ° C to obtain the test sample. The liquid phase vials containing ginsenoside Rg5 and ginsenoside Rg5a standards were placed at 25 ° C for 5, 10, 15, 20, and 25 days. The sample was dissolved in 1 mL of chromatographic 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 in the figure, after 25 days, the target peak area of ​​ginsenoside Rg5 decreased by about 90%, while the target peak area of ​​ginsenoside Rg5a decreased by less than 15%. It can be seen that the stability of the derivatives has been greatly improved.

[0063] Example 5

[0064] In this example, the anti-inflammatory effects of ginsenoside Rg5a, ginsenoside Rg5, and ginsenoside Rh4 prepared in Example 1 were analyzed by the following cell experiments.

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

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

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

[0068] Ginsenoside Rg5 group / ginsenoside Rg5a group / ginsenoside Rh4 group: Different ginsenosides with final concentrations of 10μM, 20μM, 30μM, 40μM, and 50μM were added to treat FFA-HepG2 cells, and the volume of drugs added to each group (1μL drug (or DMSO) / mL culture medium) was the same. After adding drugs and continuing to culture cells for 24 hours, the CCK8 method was used to detect cell viability. The CCK8 stock solution was diluted with serum-free MEM culture medium at a ratio of 1:10 to become a working solution, the culture medium in the 96-well plate was aspirated, and 100uL of CCK8 working solution was added again. After continuing to incubate in a 37°C incubator in the dark for 2 hours, the proliferation of FFA-HepG2 cells in the above experimental groups was detected and analyzed.

[0069] The results are as follows Figure 6 As shown in the figure, ginsenoside Rg5 and ginsenoside Rh4 groups had a certain inhibitory effect on the proliferation of FFA-HepG2 cells at a concentration of 40μM or 50μM; while ginsenoside Rg5a group had no toxic side effects on FFA-HepG2 cells at the above concentrations; it can be seen that ginsenoside Rg5a is less toxic than the other two. The concentration of 50μM was subsequently used to investigate the effects of different ginsenosides on FFA-HepG2 cells.

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

[0071] HepG2 cells in the logarithmic growth phase were inoculated into 6-well plates. After free fatty acid (FFA)-induced HepG2 non-alcoholic steatohepatitis model was established, blank group, model group, ginsenoside Rg5 group, ginsenoside Rg5a group, and ginsenoside Rh4 group were set up for treatment.

[0072] Blank group: DMSO was added;

[0073] Model group: 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 of different experimental groups was detected using a kit. Figure 7 As shown, ginsenoside Rg5a had 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 inoculated into 6-well plates. After free fatty acid (FFA)-induced HepG2 non-alcoholic steatohepatitis model was established, blank group, model group, ginsenoside Rg5 group, ginsenoside Rg5a group, and ginsenoside Rh4 group were set up for treatment.

[0078] Blank group: DMSO was added;

[0079] Model group: 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] The cells were stained with Oil Red O staining kit to observe the intracellular lipid accumulation.

[0082] The results are as follows Figure 8 As shown in the figure, it can be seen that FFA can obviously induce lipid accumulation, which is manifested by the appearance of a large number of lipid droplets in the cells of the model group, and the red area is significantly increased compared with the blank group; after the drug action, the red area decreases, reducing the lipid accumulation in the cells. Among them, after the action of ginsenoside Rg5a, the red area is the smallest, which shows that ginsenoside Rg5a has the best lipid-lowering effect.

[0083] Example 6

[0084] In this example, the anti-inflammatory effects of (R)-(-)-Rolipram, ginsenoside Rg5a prepared in Example 1, and their combination were analyzed by the following cell experiments.

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

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

[0087] Model group: 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 10 μM, 20 μM, 30 μM, 40 μM, and 50 μM, 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 ginsenoside Rg5a and (R)-(-)-Rolipram were added at a final concentration of 20 μM, or ginsenoside Rg5a and (R)-(-)-Rolipram were added at a final concentration of 30 μM, or ginsenoside Rg5a and (R)-(-)-Rolipram were added at a final concentration of 40 μM, or ginsenoside Rg5a and (R)-(-)-Rolipram were added 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 (1 μL drug (or DMSO) / mL culture medium) was the same. After adding drugs and continuing to culture cells for 24 hours, the CCK8 method was used to detect cell viability. The CCK8 stock solution was diluted with serum-free MEM culture medium at a ratio of 1:10 to become a working solution, and then the culture medium in the 96-well plate was aspirated, and 100 ul of CCK8 working solution was added again. After continuing to incubate in a 37°C incubator in the dark for 2 hours, the proliferation of FFA-HepG2 cells in the above experimental groups was detected and analyzed.

[0092] The results are as follows Fig. 9As shown, compared with the control group, ginsenoside Rg5a, (R)-(-)-Rolipram and the combination of the two had no inhibitory effect on the proliferation of FFA-HepG2 cells, indicating that the two compounds had no toxic side effects on FFA-HepG2 cells at their concentrations.

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

[0094] HepG2 cells in the logarithmic growth phase were inoculated in 6-well plates. After free fatty acid (FFA)-induced HepG2 non-alcoholic steatohepatitis model was established, blank group, model group, ginsenoside Rg5a group, (R)-(-)-Rolipram group, and ginsenoside Rg5a combined with (R)-(-)-Rolipram group were set up, and different drugs were added for treatment.

[0095] Blank group: DMSO was added;

[0096] Model group: 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 group: Ginsenoside Rg5a with a final concentration of 30 μM and (R)-(-)-Rolipram with a final concentration of 50 μM, or Ginsenoside Rg5a with a final concentration of 40 μM and (R)-(-)-Rolipram with a final concentration of 50 μM, or Ginsenoside Rg5a with a final concentration of 50 μM and (R)-(-)-Rolipram with a final concentration of 50 μM, or Ginsenoside Rg5a with a final concentration of 60 μM and (R)-(-)-Rolipram with a final concentration of 50 μM were added. The CI values ​​of different combination ratios were calculated.

[0100] The results are as follows Fig.10 The synergistic effect was the best when the molar ratio was 1:1. Subsequent experiments were conducted using 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 inoculated in 6-well plates. After free fatty acid (FFA)-induced HepG2 non-alcoholic steatohepatitis model was established, blank group, model group, ginsenoside Rg5a group, (R)-(-)-Rolipram group, and ginsenoside Rg5a combined with (R)-(-)-Rolipram group were set up, and different drugs were added for treatment.

[0103] Blank group: DMSO was added;

[0104] Model group: 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 (R)-(-)-Rolipram at a concentration of 50 μM;

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

[0108] Afterwards, the Oil Red O staining kit was used to detect the intracellular lipid accumulation; the ELISA test kit was used to detect the contents of IL-6 and TNF-α in different experimental groups.

[0109] The results are as follows Fig.11 , Fig.12 As shown, from Fig.11 It can be seen that after modeling, the cells had obvious lipid accumulation. After the drug action, the degree of cell lipid accumulation was reduced; in the combined drug group, the cell lipid accumulation was significantly reduced. Fig.12 It can be seen that the secretion of IL-6 and TNF-α by cells in the model group was significantly higher than that in the blank control group. After intervention in the combined medication group, the secretion of the above inflammatory factors was significantly reduced.

[0110] Example 7 Animal Experiment

[0111] High-fat and high-sugar HFHC diet induced non-alcoholic fatty liver disease in mice: 6-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 with ordinary feed. The other three groups were fed with D12492 high-fat feed and given 4% sugar water (containing 45% sucrose and 55% fructose) for 8 consecutive weeks to establish a non-alcoholic fatty liver disease model. After 8 weeks, the drug-treated groups were intraperitoneally injected for 4 weeks (the dose of ginsenoside Rg5a was 40 mg / kg, the dose of (R)-(-)-Rolipram group was 14 mg / kg, and the dose of ginsenoside Rg5a combined with (R)-(-)-Rolipram group was ginsenoside Rg5a 20 mg / kg + (R)-(-)-Rolipram 7 mg / kg).

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

[0113] After freezing the liver tissue, cut the frozen tissue and attach it to the slide, and return to room temperature. After soaking in distilled water, rinse with 60% isopropanol, then put it in the freshly prepared Oil Red O staining solution, and rinse with distilled water. Seal with water-soluble mounting medium and use neutral gum to seal the edge of the slide and cover glass.

[0114] Oil Red O is a fat-soluble dye that can color neutral fats such as triglycerides in tissues. Therefore, the lipid droplets in tissue cells appear red and the cell nuclei appear blue. The proportion of red area in the slice can reflect the severity of lipid accumulation. Fig.13 As shown, the red area in the model group accounted for the largest proportion, while the red area in the combined medication group was significantly reduced and the degree of lipid accumulation was alleviated.

[0115] 2. H&E staining of liver tissue:

[0116] The liver tissue sections after hydration were soaked in pure water, stained with hematoxylin, and rinsed with running water. Then, they were differentiated, rinsed, blued, and rinsed. After the water was controlled, they were stained with eosin solution for 30 seconds, rinsed, dried, sealed with neutral gum, examined under a microscope, and imaged for analysis.

[0117] H&E staining can reflect the degree of liver lesions. Fig.14 As shown, in the model group, central vein dilation, widened hepatic cords, disordered arrangement, partial hepatic sinus congestion, and unclear hepatocyte boundaries were observed. After combined drug treatment, the structure of the hepatic lobule tended to return to normal, necrotic cells were significantly reduced, and fat vacuoles were reduced, which significantly improved the degree of liver lesions.

[0118] 3. Masson staining of liver tissue

[0119] After paraffin sections were dewaxed to water, they were stained with hematoxylin for 5 minutes, then differentiated, rinsed with distilled water, blued, counterstained with Ponceau red, rinsed, and counterstained with aniline blue. They were then immediately placed in 1% glacial acetic acid for dehydration, transparentized, and sealed with neutral gum.

[0120] Masson staining can be used to identify collagen fibers and muscle fibers. In the staining results, collagen fibers appear blue and muscle fibers appear red. Fig.15 As shown in the figure, the normal structure of the liver lobules in the normal group existed, while Masson trichrome staining of the liver portal area in the model group showed thick dark blue fibers, indicating liver fibrosis changes, and fatty degeneration of hepatocytes could be observed. After combined drug treatment, the blue staining area was significantly reduced, and the distribution range was limited to the periphery of the vascular wall, indicating that the degree of liver fibrosis can be significantly improved.

[0121] 4. Cholesterol CHO and triglyceride TG content in mouse serum

[0122] In order to explore the effect of drug action on lipid abnormalities caused by non-alcoholic steatohepatitis, the expression levels of cholesterol CHO and TG in the serum of mice were evaluated.

[0123] The level of TG in the liver can directly reflect the lipid-lowering effect of the drug. Fig.16 As shown in the figure, a represents ginsenoside Rg5a, and b represents (R)-(-)-Rolipram. The CHO and TG contents in the model group were significantly increased compared with the blank group. Combined drug treatment can reduce the CHO and TG levels in the mouse serum to normal levels.

[0124] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent structural change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A ginsenoside Rg5 derivative, characterized in that: It has the following structural formula: The molecular formula is: C 42 H 74 O 12 ; Molecular weight: 770.5180.

2. A method for synthesizing the ginsenoside Rg5 derivative as claimed in claim 1, characterized in that: The following steps are involved: Step 1, dissolving ginsenoside Rg5 in anhydrous ethanol, then adding palladium carbon catalyst, and then adding an aqueous solution of sodium hypophosphite monohydrate, reacting at room temperature, filtering to remove the palladium carbon catalyst after the reaction is completed, extracting with dichloromethane, washing the organic phase with saturated brine, drying with anhydrous sodium sulfate, filtering and concentrating under reduced pressure to obtain a crude product; Step 2: dissolve the crude product described in step 1 in anhydrous ethanol, purify it by semi-preparative liquid chromatography, collect the eluate of ginsenoside Rg5 derivative, and dry the collected eluate by rotary evaporation to obtain the product ginsenoside Rg5 derivative.

3. The method according to claim 2, characterized in that In step 1, the ratio of the mass of ginsenoside Rg5 to the volume of anhydrous ethanol is 1:(4-8), wherein the unit of mass is g and the unit of volume is mL; the mass of the palladium-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.

4. The method according to claim 2, characterized in that: The reaction time in step 1 is 15 h to 30 h.

5. The method according to claim 2, characterized in that: The separation conditions of the semi-preparative liquid chromatography in step 2 are: C18 column (20×250 mm, 10 μm), UV detector, detection wavelength of 203 nm, volume flow rate of 10 mL / min, mobile phase A is pure water, B is acetonitrile, gradient elution program: 0-100 min, 45%-55% acetonitrile.

6. Use of the ginsenoside Rg5 derivative as claimed in claim 1 in the preparation of a drug for treating non-alcoholic fatty hepatitis.

7. The use according to claim 6, characterized in that: The medicine includes ginsenoside Rg5 derivatives.

8. The use according to claim 7, characterized in that: The medicaments also include PDE4 inhibitors.

9. The use according to claim 8, characterized in that: The PDE4 inhibitor is (R)-(-)-Rolipram.

10. The use according to claim 9, characterized in that: The molar ratio of the ginsenoside Rg5 derivative to (R)-(-)-Rolipram in the drug is 1:1.

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