Glycoside eutectic composition and application thereof in treatment of metabolism-related fatty liver disease

Abstract

The invention provides a glycoside eutectic composition and application thereof in treatment of metabolism-related fatty liver diseases, at present, clinical drugs for MAFLD are extremely few and have great side effects, the glycoside composition which has good treatment / prevention effects on MAFLD is prepared through a molecular compound strategy by referring to the overall view of the theory of traditional Chinese medicine, the synergistic interaction effect of the glycoside composition is achieved, and the application of the glycoside eutectic composition in treatment of metabolism-related fatty liver diseases is achieved. And the compound has a certain protection effect on the liver function of the MAFLD mouse. However, many active glycoside compounds are insoluble in water and low in bioavailability, and the glycoside compound eutectic crystal provided by the invention further improves the effect of preventing and treating MAFLD.

Description

Technical Field

[0001] The present invention belongs to the technical field of functional component screening and application, and specifically relates to a glycoside cocrystal composition and its application in treating metabolism-related fatty liver disease. Background Art

[0002] Metabolic dysfunction-Associated Fatty Liver Disease (MAFLD), formerly known as non-alcoholic fatty liver disease (NAFLD), is a chronic liver disease associated with metabolic dysfunction, with abnormal accumulation of liver fat as the main pathological change. With the increase of bad living habits such as staying up late and sitting for long periods of time, and unhealthy eating habits such as high calories, high fat, and high sugar, its incidence rate is becoming popular worldwide. According to epidemiological surveys, the incidence rate of metabolic-associated fatty liver disease in my country is also increasing year by year, and it is showing a trend of younger people.

[0003] Modern medicine mainly adopts methods such as adjusting diet structure, controlling weight, anti-inflammatory and lipid regulation, lowering enzymes and protecting the liver, and improving insulin resistance to prevent and treat MAFLD. For decades, only the new drug Resmetirom developed by Madrigal passed FDA approval in the first half of 2024, but the drug was found to have obvious side effects in the clinical stage, including uncontrollable abnormal liver function. Therefore, it is particularly urgent to continue to find new candidate drugs with low toxicity and high efficiency for the prevention and treatment of MAFLD. Summary of the invention

[0004] The present invention provides a glycoside cocrystal composition and application thereof in treating metabolism-related fatty liver disease, thereby making up for the deficiencies of the prior art.

[0005] The present invention first provides a composition, which is a composition of luteolin and oleuropein, a composition of luteolin and baicalin, or a composition of astragaloside IV, luteolin-4'-O-glucoside and glycyrrhizic acid;

[0006] The luteolin and oleuropein composition, wherein the mass ratio of luteolin to oleuropein is 1:1;

[0007] The composition of luteolin and baicalin, wherein the mass ratio of luteolin to baicalin is 5:1;

[0008] The mass ratio of astragaloside IV, luteolin-4'-O-glucoside and glycyrrhizic acid is 1:1:10.

[0009] Furthermore, the composition further comprises a composition of luteolin, oleuropein and atorvastatin in a mass ratio of 50:50:1;

[0010] The composition further comprises oleuropein-nicotinamide cocrystal and luteolin-lysine cocrystal composition in a mass ratio of 1:1,

[0011] The oleuropein-nicotinamide co-crystal is prepared by placing oleuropein and nicotinamide in a ball mill, adding drops of anhydrous ethanol and grinding for h, and drying the obtained powder in a vacuum dryer to prepare the oleuropein-nicotinamide co-crystal;

[0012] The preparation method of the luteolin-lysine cocrystal comprises the following steps:

[0013] 0.1 mmol of luteolin and 0.1 mmol of lysine were placed in a ball mill, 3 drops of anhydrous ethanol were added, and the mixture was ground at 1200 r / min for 2 h. The obtained powder was dried in a vacuum drying oven for 12 h to obtain luteolin-lysine cocrystal.

[0014] The composition further comprises a composition of oleuropein-nicotinamide cocrystal, luteolin-lysine cocrystal and polypeptide in a mass ratio of 1:1:1;

[0015] The amino acid sequence of the polypeptide is NCASLQSKMLMFDY (SEQ ID NO: 1).

[0016] The composition of the present invention can be used to prepare a medicament for preventing and / or treating liver diseases.

[0017] The present invention provides a glycoside composition with good treatment / prevention of MAFLD, achieves its synergistic effect, and has a protective effect on the liver function of MAFLD mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a diagram of the establishment of a high-fat model in normal human hepatocytes (L02).

[0019] Figure 2 This is a diagram of the establishment of a high glucose model for normal human hepatocytes (L02).

[0020] Figure 3 This is a diagram of the establishment of the human normal hepatocyte (L02) inflammation model.

[0021] Figure 4 This is a human normal hepatocyte (L02) inflammation model: the left picture is the blank control group, the middle picture is the model group, and the right picture is the Example 7 group. DETAILED DESCRIPTION

[0022] The method for establishing the high-fat model of normal human liver cells (L02) in the embodiment is as follows:

[0023] L02 cells were seeded in 96-well plates (0.5×10 4 cells / well, 180 μL per well), after culturing for 24 hours, discard the old culture medium, wash once with PBS, add 20 μL culture medium to the control group, and add 20 μL palmitic acid of different concentration gradients (final concentrations are 0.05, 0.075, 0.1, 0.125, 0.15, 0.175, 0.2, 0.3 and 0.4 mM) to the experimental group and continue to culture for 24 hours. After the culture is completed, add 20 μL of MTT (5 mg / mL) solution to each well under light-proof conditions and continue to incubate for 3 hours, discard the supernatant, add 150 μL DMSO to each well, shake for 10 minutes, and measure the absorbance (OD value) at 492 nm. Set up 6 duplicate wells in each group, repeat 3 times, and the results are expressed as cell survival rate ( Figure 1 ).

[0024] The method for establishing a high glucose model of normal human liver cells (L02) in the embodiment of the present invention is as follows:

[0025] L02 cells were seeded in 96-well plates (0.5×10 4 cells / well, 180 μL per well), after culturing for 24 h, discard the old culture medium, wash once with PBS, add 20 μL culture medium to the control group, and add 180 μL of glucose with different concentration gradients (final concentrations were 100, 150, 200, 250, 300, 350, 400, 450, 500 mM, respectively) to the experimental group, model for 24 h, add 20 μL of MTT (5 mg / mL) solution to each well under light-proof conditions and continue incubation for 3 h, discard the supernatant, add 150 μL DMSO to each well, shake for 10 min, and measure the absorbance (OD value) at 492 nm. Set 6 duplicate wells in each group, repeat 3 times, and the results are expressed as cell survival rate ( Figure 2 ).

[0026] The method for establishing the inflammation model of normal human liver cells (L02) in the embodiment of the present invention is as follows:

[0027] L02 cells were seeded in 96-well plates (0.5×10 4cells / well, 180 μL per well), after culturing for 24 hours, discard the old culture medium, wash once with PBS, add 20 μL culture medium to the control group, and add 180 μL of lipopolysaccharide LPS of different concentration gradients (final concentrations are 0.1, 0.5, 1, 2, 5, 10 μg / mL) to the experimental group, model for 24 hours, add 20 μL of MTT (5 mg / mL) solution to each well under light-proof conditions and continue incubation for 3 hours, discard the supernatant, add 150 μL DMSO to each well, shake for 10 minutes, and measure the absorbance (OD value) at 492 nm. Set up 6 duplicate wells in each group and repeat 3 times. The results are expressed as cell survival rate ( Figure 3 ).

[0028] The steps of evaluating the MAFLD prevention and treatment activity of the embodiment using the L02 cell high lipid model are as follows:

[0029] L02 cells were exposed to 0.125mM palmitic acid solution for 24h to simulate a high-fat state, and treated with different concentration gradient samples. After treatment, the survival rate of L02 cells was determined by MTT method, and the morphology of L02 cells was observed using an inverted microscope. The total cholesterol (TC) and total triglyceride (TG) content of cells were detected using the kit method. 6 replicate wells were repeated 3 times.

[0030] The steps of evaluating the MAFLD prevention and treatment activity of the embodiment using the L02 cell high glucose model in the present invention are as follows:

[0031] L02 cells were exposed to 200mM glucose solution for 24h to simulate high glucose state, and treated with different concentration gradient samples. After treatment, the survival rate of L02 cells was determined by MTT method, and the morphology of L02 cells was observed using an inverted microscope. The content of cellular inflammatory factors IL-6 (interleukin-6), TNFα (tumor necrosis factor α), and NF-κB (nuclear factor κB) was detected using the kit method. 6 duplicate wells, repeated 3 times.

[0032] The steps of evaluating the activity of the embodiment for preventing and treating MAFLD using the L02 cell inflammation model are as follows:

[0033] L02 cells were exposed to 1 μg / mL LPS solution for 10 h to simulate the inflammatory state, and treated with samples of different concentration gradients. After treatment, the survival rate of L02 cells was determined by the MTT method, and the morphology of L02 cells was observed using an inverted microscope. The content of cellular inflammatory factors IL-6 (interleukin-6), TNFα (tumor necrosis factor α), and NF-κB (nuclear factor κB) was detected using the kit method. 6 duplicate wells were repeated 3 times. The ROS content in the cells was tested using a ROS fluorescent probe ( Figure 4 ).

[0034] The steps of evaluating the prevention and treatment of MAFLD and liver function protection effects of the embodiments using the mouse MAFLD model are as follows:

[0035] Take 82 male Kunming mice, weighing 18-22g, and randomly divided into a control group, a model group, and an embodiment 1-7 group according to body weight after adaptive feeding. Except that the control group is given ordinary feed, the remaining groups are all given MCD (methionine choline deficiency feed) for 1 month. At the same time, the drug administration group is gavaged once a day, and the administration volume is 10mL / kg, and the drug administration is continued for 1 month, and the control group is given an equal volume of solvent gavage. After the last administration, the mice are fasted but not water-restricted. After 14 hours, serum is taken to detect mouse liver function indexes AST (aspartate aminotransferase), ALT (alanine aminotransferase), TBA (total bile acid) and blood lipid-related indexes TC (total cholesterol), TG (total triglycerides) using a kit method and calculate liver index.

[0036] The effects of the compositions in different embodiments were tested according to the above models and indicators.

[0037] Example 1

[0038] A composition of luteolin and oleuropein in a mass ratio of 1:1.

[0039] Example 2

[0040] A composition of luteolin and baicalin in a mass ratio of 5:1.

[0041] Example 3

[0042] A composition of astragaloside IV, luteolin-4'-O-glucoside and glycyrrhizic acid in a mass ratio of 1:1:10.

[0043] The detection results of the above three compositions are shown in the following table.

[0044] Table 1: Evaluation of the activity of glycoside combination in preventing and treating MAFLD using L02 cell hyperlipidemia model

[0045]

[0046] As shown in Table 1, for the L02 cell hyperlipidemia model, the cell survival rates of all embodiments were higher than those of the model group, and the TC and TG contents were lower than those of the model group; the cell survival rate of Example 1 was the highest, and the TC and TG contents were the lowest, indicating that the composition of luteolin and oleuropein with a mass ratio of 1:1 had a better protective effect on L02 hyperlipidemia cells.

[0047] Table 2: Evaluation of the activity of glycoside combination in preventing and treating MAFLD using L02 cell high glucose model

[0048]

[0049]

[0050] As shown in Table 2, for the L02 cell high glucose model, the cell survival rates of all embodiments were higher than those of the model group, and the IL-6 and TNFα contents were lower than those of the model group; the cell survival rate of Example 1 was the highest, and the IL-6 and TNFα contents were the lowest, indicating that the composition of luteolin and oleuropein with a mass ratio of 1:1 had a better protective effect on L02 high glucose cells.

[0051] Table 3: Evaluation of the activity of glycoside composition in preventing and treating MAFLD using L02 cell inflammation model

[0052]

[0053] As shown in Table 3, for the L02 cell inflammation model, the cell survival rates of all embodiments were higher than those of the model group, and the IL-6 and TNFα contents were lower than those of the model group; the cell survival rate of Example 1 was the highest, and the IL-6 and TNFα contents were the lowest, indicating that the composition of luteolin and oleuropein with a mass ratio of 1:1 had a better protective effect on L02 inflammatory cells.

[0054] Table 4: Evaluation of the activity of different glycoside compositions in preventing and treating MAFLD in mouse MAFLD model

[0055]

[0056]

[0057] As can be seen from Table 4, for the mouse MAFLD model: in terms of liver index, the liver index of mice in all embodiments was significantly lower than that in the model group, and the liver index of Example 1 was the lowest; in terms of liver function, compared with the model group, the TBA, ALT and AST of mice in all embodiments were significantly reduced, and the TBA, ALT and AST of Example 1 were the lowest; in terms of blood lipids, compared with the model group, all embodiments could reduce the TG and TG content in mouse serum, and the TC and TG of Example 1 were the lowest, indicating that oleuropein and oleuropein in a mass ratio of 1:1 have a certain therapeutic effect on MAFLD in mice, and have a certain protective effect on the liver function of mice.

[0058] Based on the above experimental results, the compositions of Examples 4 to 7 are provided for verification.

[0059] Example 4

[0060] A composition of luteolin, oleuropein and licorice extract in a mass ratio of 1:1:100.

[0061] Example 5

[0062] A composition of luteolin, oleuropein and atorvastatin in a mass ratio of 50:50:1.

[0063] Example 6

[0064] A composition of oleuropein-nicotinamide cocrystal and luteolin-lysine cocrystal in a mass ratio of 1:1.

[0065] The preparation method of oleuropein-nicotinamide cocrystal comprises the following steps:

[0066] 0.1 mmol oleuropein and 0.4 mmol nicotinamide were placed in a ball mill, 3 drops of anhydrous ethanol were added, and the mixture was ground at 1200 r / min for 2 h. The obtained powder was dried in a vacuum drying oven for 12 h to obtain oleuropein-nicotinamide cocrystal.

[0067] The preparation method of luteolin-lysine cocrystal comprises the following steps:

[0068] 0.1 mmol of luteolin and 0.1 mmol of lysine were placed in a ball mill, 3 drops of anhydrous ethanol were added, and the mixture was ground at 1200 r / min for 2 h. The obtained powder was dried in a vacuum drying oven for 12 h to obtain luteolin-lysine cocrystal.

[0069] Example 7

[0070] Oleuropein-nicotinamide cocrystal, luteolin-lysine cocrystal and polypeptide composition with a mass ratio of 1:1:1.

[0071] The method for preparing the polypeptide comprises the following steps:

[0072] The de-oiled olive pomace was mixed with distilled water at a solid-liquid ratio of 1:30 (g / ml), and the pH was adjusted to 9.0 with 0.1mol / L NaOH. The supernatant was centrifuged and the precipitate was extracted twice, and the supernatant was combined. The pH was adjusted to 4.0 with 0.1mol / L HCI solution. The protein precipitate was collected by centrifugation, washed to neutrality, and freeze-dried to obtain olive pomace protein. Olive pomace protein was mixed with distilled water at a solid-liquid ratio of 1:10 (g / ml), homogenized with a high-speed shearing machine for 30 minutes, 3.0g papain was added to the homogenate and hydrolyzed at 60℃ for 2h, and the hydrolyzate was centrifuged, decolorized, concentrated, and dried to obtain polypeptides. Active polypeptides were screened by MAFLD activity identification.

[0073] Through mass spectrometry identification, the amino acid sequence of one of the polypeptide fragments with better activity was NCASLQSKMLMFDY, and its MAFLD activity was as follows: liver index 513±22, ALT (U / L) 195±35, AST (U / L) 438±29, TBA (μmol / L) 20±5, TG (mmol / L) 0.7±0.3, TC (mmol / L) 3.7±0.3.

[0074] The effects of each composition of Example 4 to Example 7 are shown in the following table.

[0075] Table 5: Evaluation of the activity of glycoside combination in preventing and treating MAFLD using L02 cell hyperlipidemia model

[0076]

[0077]

[0078] As shown in Table 5, for the L02 cell hyperlipidemia model, the cell survival rates of all examples were higher than those of the model group, and the TC and TG contents were lower than those of the model group; Example 7 had the highest cell survival rate and the lowest TC and TG contents, indicating that the glycoside cocrystal composition has a good protective effect on L02 hyperlipidemia cells, and the effect of the glycoside cocrystal and polypeptide composition is better.

[0079] Table 6: Evaluation of the activity of glycoside combination in preventing and treating MAFLD using L02 cell high glucose model

[0080]

[0081] As can be seen from Table 6, for the L02 cell high-glucose model, the cell survival rates of all embodiments were higher than those of the model group, and the IL-6 and TNFα contents were lower than those of the model group; Example 7 had the highest cell survival rate, and the lowest IL-6 and TNFα contents, indicating that the glycoside co-crystal composition has a good protective effect on L02 high-glucose cells, and the effect of the glycoside co-crystal and polypeptide composition is better.

[0082] Table 7: Evaluation of the activity of glycoside composition in preventing and treating MAFLD using L02 cell inflammation model

[0083]

[0084] As can be seen from Table 7, for the L02 cell inflammation model, the cell survival rates of all embodiments were higher than those of the model group, and the IL-6 and TNFα contents were lower than those of the model group; Example 7 had the highest cell survival rate, and the lowest IL-6 and TNFα contents, indicating that the glycoside co-crystal composition has a good protective effect on L02 inflammatory cells, and the effect of the glycoside co-crystal and polypeptide composition is better.

[0085] Table 8: Evaluation of the activity of different glycoside compositions in preventing and treating MAFLD using the mouse MAFLD model

[0086]

[0087] As can be seen from Table 8, for the mouse MAFLD model: in terms of liver index, compared with the model group, the liver index of mice in all embodiments was significantly reduced, and the liver index of Example 7 was the lowest; in terms of liver function, compared with the model group, the TBA, ALT and AST of mice in all embodiments were significantly reduced, and the TBA, ALT and AST of Example 7 were the lowest; in terms of blood lipids, compared with the model group, all embodiments can reduce the TG and TG content in mouse serum, and the TC and TG of Example 7 are the lowest, indicating that the glycoside co-crystal composition has a certain therapeutic effect on MAFLD in mice, and has a certain protective effect on the liver function of mice. Furthermore, the effect of the glycoside co-crystal and polypeptide composition is better.

[0088] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that several improvements and modifications may be made without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A composition, characterized in that The composition is a composition of luteolin and oleuropein, a composition of luteolin and baicalin, or a composition of astragaloside IV, luteolin-4'-O-glucoside and glycyrrhizic acid.

2. The composition according to claim 1, characterized in that In the luteolin and oleuropein composition, the mass ratio of luteolin to oleuropein is 1:

1.

3. The composition according to claim 1, characterized in that The mass ratio of luteolin and baicalin in the luteolin and baicalin composition is 5:

1.

4. The composition according to claim 1, characterized in that The mass ratio of astragaloside IV, luteolin-4'-O-glucoside and glycyrrhizic acid is 1:1:

10.

5. The composition according to claim 1, characterized in that The composition also comprises a composition of luteolin, oleuropein and atorvastatin in a mass ratio of 50:50:

1.

6. The composition according to claim 1, characterized in that The composition further comprises a composition of oleuropein-nicotinamide cocrystal and luteolin-lysine cocrystal in a mass ratio of 1:1, The oleuropein-nicotinamide co-crystal is prepared by placing oleuropein and nicotinamide in a ball mill, adding drops of anhydrous ethanol and grinding for h, and drying the obtained powder in a vacuum dryer to prepare the oleuropein-nicotinamide co-crystal; The preparation method of the luteolin-lysine cocrystal comprises the following steps: 0.1 mmol of luteolin and 0.1 mmol of lysine were placed in a ball mill, 3 drops of anhydrous ethanol were added, and the mixture was ground at 1200 r / min for 2 h. The obtained powder was dried in a vacuum drying oven for 12 h to obtain luteolin-lysine cocrystal.

7. The composition according to claim 1, characterized in that The composition further comprises a composition of oleuropein-nicotinamide cocrystal, luteolin-lysine cocrystal and a polypeptide in a mass ratio of 1:1:1; the amino acid sequence of the polypeptide is SEQ ID NO:

1.

8. A polypeptide having the efficacy of preventing and / or treating liver diseases, characterized in that: The amino acid sequence of the polypeptide is SEQ ID NO:

1.

9. Use of the composition according to any one of claims 1 to 7 in the preparation of a medicament for preventing and / or treating liver diseases.

10. A drug for preventing and / or treating liver diseases, characterized in that: The medicine contains the composition according to any one of claims 1 to 7 and / or the polypeptide according to claim 8 at a pharmacologically effective concentration.

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