A glycoside cocrystal composition and its application in the treatment of metabolic-associated fatty liver disease

Abstract

This invention provides a glycoside cocrystal composition and its application in the treatment of metabolic-associated fatty liver disease (MAFLD). Currently, there are very few clinical drugs for MAFLD, and they often have significant side effects. This invention draws on the holistic view of traditional Chinese medicine and uses a molecular compound strategy to prepare a glycoside composition with good therapeutic / preventive effects on MAFLD, achieving a synergistic effect and providing some protection against liver function in MAFLD mice. However, many active glycoside compounds are poorly soluble in water and have low bioavailability. The glycoside cocrystal provided by this invention further enhances its efficacy in preventing and treating MAFLD.

Description

Technical Field

[0001] This invention belongs to the field of functional component screening and application technology, specifically relating to a glycoside cocrystal composition and its application in the treatment of metabolic-related fatty liver disease. Background Technology

[0002] Metabolic dysfunction-associated fatty liver disease (MAFLD), formerly known as non-alcoholic fatty liver disease (NAFLD), is a chronic liver disease characterized by abnormal fat accumulation in the liver, associated with metabolic dysfunction. With the increasing prevalence of unhealthy lifestyle habits such as staying up late and prolonged sitting, as well as unhealthy dietary habits involving high-calorie, high-fat, and high-sugar foods, its incidence is showing a global trend. Epidemiological surveys show that the incidence of MAFLD in my country is also increasing year by year, and is showing a trend towards affecting younger people.

[0003] Modern medicine primarily addresses MAFLD through dietary adjustments, weight control, anti-inflammatory and lipid-regulating measures, liver protection by lowering enzymes, and improving insulin resistance. Over the past decades, only Resmetirom, developed by Madrigal, received FDA approval in the first half of 2024; however, significant side effects were observed during clinical trials, including uncontrollable liver dysfunction. Therefore, the search for new, low-toxicity, and highly effective drug candidates for the prevention and treatment of MAFLD is particularly urgent. Summary of the Invention

[0004] This invention provides a glycoside cocrystal composition and its application in the treatment of metabolic-related fatty liver disease, thereby overcoming the shortcomings of the prior art.

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

[0006] The composition of luteolin and oleuropein, wherein the mass ratio of luteolin and 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 A, luteolin-4'-O-glucoside and glycyrrhizic acid is 1:1:10.

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

[0010] The composition further comprises a cocrystal of oleuropein-nicotinamide and a cocrystal of luteolin-lysine 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 dropwise anhydrous ethanol, grinding for h, and then drying the resulting powder in a vacuum dryer.

[0012] The preparation method of the luteolin-lysine co-crystal includes 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 resulting 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 polypeptide described herein has the amino acid sequence NCASLQSKMLMFDY (SEQ ID NO:1).

[0016] The compositions of the present invention can be used to prepare medicaments for the prevention and / or treatment of liver diseases.

[0017] This invention provides a glycoside composition with good therapeutic / preventive effects for MAFLD, achieving a synergistic effect and protecting liver function in MAFLD mice. Attached Figure Description

[0018] Figure 1 This diagram illustrates the establishment of a hyperlipidemia model in normal human hepatocytes (L02).

[0019] Figure 2 Diagram showing the establishment of a high-glucose model in normal human hepatocytes (L02).

[0020] Figure 3 Diagram showing the establishment of an inflammation model of normal human hepatocytes (L02).

[0021] Figure 4 Human normal hepatocyte (L02) inflammation model: the left figure is the blank control group, the middle figure is the model group, and the right figure is the Example 7 group. Detailed Implementation

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

[0023] L02 cells were seeded in 96-well plates (0.5 × 10⁻⁶ cells per well). 4 Cells per well (180 μL per well) were cultured for 24 h. Afterward, the old culture medium was discarded, and the cells were washed once with PBS. The control group received 20 μL of culture medium, while the experimental groups received 20 μL of palmitic acid at different concentration gradients (final concentrations of 0.05, 0.075, 0.1, 0.125, 0.15, 0.175, 0.2, 0.3, and 0.4 mM), and were cultured for another 24 h. After culture, 20 μL of MTT (5 mg / mL) solution was added to each well under dark conditions, and the cells were incubated for another 3 h. The supernatant was discarded, and 150 μL of LDMSO was added to each well. The cells were shaken for 10 min, and the absorbance (OD value) at 492 nm was measured. Each group had 6 replicates, repeated 3 times. Results were expressed as cell viability. Figure 1 ).

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

[0025] L02 cells were seeded in 96-well plates (0.5 × 10⁻⁶ cells per well). 4 Cells per well, 180 μL per well. After culturing for 24 h, the old culture medium was discarded, and the cells were washed once with PBS. The control group was given 20 μL of culture medium, while the experimental groups were given 180 μL of glucose at different concentration gradients (final concentrations of 100, 150, 200, 250, 300, 350, 400, 450, and 500 mM). After 24 h of modeling, 20 μL of MTT (5 mg / mL) solution was added to each well under dark conditions, and incubation continued for 3 h. The supernatant was discarded, and 150 μL of DMSO was added to each well. The cells were shaken for 10 min, and the absorbance (OD value) at 492 nm was measured. Each group had 6 replicates, repeated 3 times. Results were expressed as cell viability. Figure 2 ).

[0026] The method for establishing an inflammation model using normal human hepatocytes (L02) in this embodiment of the invention is as follows:

[0027] L02 cells were seeded in 96-well plates (0.5 × 10⁻⁶ cells per well). 4Cells per well (180 μL per well) were cultured for 24 h. Afterward, the old culture medium was discarded, and the cells were washed once with PBS. The control group received 20 μL of culture medium, while the experimental groups received 180 μL of different concentration gradients of lipopolysaccharide (LPS) (final concentrations of 0.1, 0.5, 1, 2, 5, and 10 μg / mL). After 24 h of incubation, 20 μL of MTT (5 mg / mL) solution was added to each well under dark conditions for another 3 h of incubation. The supernatant was discarded, and 150 μL of DMSO was added to each well. The cells were shaken for 10 min, and the absorbance (OD value) at 492 nm was measured. Each group had 6 replicates, repeated 3 times. Results are expressed as cell viability. Figure 3 ).

[0028] The steps for evaluating the anti-MAFLD activity of the L02 cell hyperlipidemia model in this invention are as follows:

[0029] L02 cells were exposed to 0.125 mM palmitic acid solution for 24 h to simulate a high-lipid state, and were simultaneously treated with samples of different concentration gradients. After treatment, the viability of L02 cells was determined by the MTT assay, and the morphology of L02 cells was observed using an inverted microscope. The levels of total cholesterol (TC) and total triglycerides (TG) in the cells were detected using a kit-based method. Six replicates were performed, with three repetitions.

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

[0031] L02 cells were exposed to 200 mM glucose solution for 24 h to simulate a high glucose state, and simultaneously treated with samples of different concentration gradients. After treatment, the viability of L02 cells was determined by the MTT assay, and the morphology of L02 cells was observed using an inverted microscope. The levels of cellular inflammatory factors IL-6 (interleukin-6), TNFα (tumor necrosis factor-α), and NF-κB (nuclear factor-κB) were detected using a kit-based method. Six replicates were performed, with three repetitions per well.

[0032] The steps for evaluating the MAFLD prevention and treatment activity of the L02 cell inflammation model in this invention are as follows:

[0033] L02 cells were exposed to 1 μg / mL LPS solution for 10 h to simulate an inflammatory state, and were simultaneously treated with samples of different concentration gradients. After treatment, the viability of L02 cells was determined by the MTT assay, and the morphology of L02 cells was observed using an inverted microscope. The levels of cellular inflammatory factors IL-6 (interleukin-6), TNFα (tumor necrosis factor-α), and NF-κB (nuclear factor-κB) were detected using a kit-based method. Six replicates were performed, with three repetitions. The ROS content in the cells was measured using a ROS fluorescent probe. Figure 4 ).

[0034] The steps for evaluating the preventive and protective effects of the embodiments on MAFLD and liver function in this invention using a mouse MAFLD model are as follows:

[0035] Eighty-two male Kunming mice, weighing 18-22g, were randomly divided into a control group, a model group, and groups according to their weight after acclimatization. Except for the control group, which received a normal diet, all other groups were given MCD (methionine-choline deficient diet) for one month. Simultaneously, the treatment groups received the drug once daily by gavage at a volume of 10mL / kg for one month, while the control group received an equal volume of solvent by gavage. After the last administration, the mice were fasted but allowed free access to water. Serum samples were collected 14 hours later, and liver function indicators AST (aspartate aminotransferase), ALT (alanine aminotransferase), TBA (total bile acids), and lipid-related indicators TC (total cholesterol) and TG (total triglycerides) were measured using a kit method. Liver index was also calculated.

[0036] The effects of the compositions in different embodiments were tested according to the above model 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 A, 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 table below.

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

[0045]

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

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

[0048]

[0049]

[0050] As shown in Table 2, for the L02 cell hyperglycemia model, the cell survival rate of all examples was higher than that of the model group, while the IL-6 and TNFα contents were lower than those of the model group. Example 1 showed the highest cell survival rate and the lowest IL-6 and TNFα contents, indicating that the luteolin and oleuropein combination with a mass ratio of 1:1 has a better protective effect on L02 hyperglycemia cells.

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

[0052]

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

[0054] Table 4: Evaluation of the anti-MAFLD activity of different glycoside compositions in a mouse MAFLD model

[0055]

[0056]

[0057] As shown in Table 4, for the mouse MAFLD model: regarding liver index, the liver index of mice in all examples was significantly lower than that of the model group, with the lowest liver index in Example 1; regarding liver function, compared with the model group, the TBA, ALT, and AST levels of mice in all examples were significantly reduced, with the lowest TBA, ALT, and AST levels in Example 1; regarding blood lipids, compared with the model group, all examples reduced the levels of TG and TG in mouse serum, with the lowest TC and TG levels in Example 1. This indicates that luteolin and oleuropein in a 1:1 mass ratio have a certain therapeutic effect on MAFLD in mice and a certain protective effect on liver function in 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 includes the following steps:

[0066] 0.1 mmol of oleuropein and 0.4 mmol of 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 resulting powder was dried in a vacuum drying oven for 12 h to obtain oleuropein-nicotinamide cocrystal.

[0067] The preparation method of luteolin-lysine co-crystal includes 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 resulting powder was dried in a vacuum drying oven for 12 h to obtain luteolin-lysine cocrystal.

[0069] Example 7

[0070] A composition of oleuropein-nicotinamide cocrystal, luteolin-lysine cocrystal, and polypeptide in a mass ratio of 1:1:1.

[0071] The preparation method of polypeptides includes the following steps:

[0072] Olive pomace (after oil removal) was mixed with distilled water at a ratio of 1:30 (g / ml). The pH was adjusted to 9.0 with 0.1 mol / L NaOH. The mixture was ultrasonicated at 300 W, 50 °C, and 1 h. The supernatant was collected by centrifugation. The precipitate was extracted twice more. The supernatants were combined, and the pH was adjusted to 4.0 with 0.1 mol / L HCl solution. The protein precipitate was collected by centrifugation, washed until neutral, and then freeze-dried to obtain olive pomace protein. Olive pomace protein was mixed with distilled water at a ratio of 1:10 (g / ml). The mixture was homogenized using a high-speed shear mill for 30 min. 3.0 g of papain was added to the homogenate, and the homogenate was enzymatically hydrolyzed at 60 °C for 2 h. The hydrolysate was centrifuged, decolorized, concentrated, and dried to obtain peptides. Active peptides were screened using MAFLD activity identification.

[0073] Mass spectrometry analysis revealed that the amino acid sequence of one of the polypeptide fragments with good 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 the compositions in Examples 4 to 7 are shown in the table below.

[0075] Table 5: Evaluation of the activity of glycoside composition in preventing MAFLD using the L02 cell hyperlipidemia model.

[0076]

[0077]

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

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

[0080]

[0081] As shown in Table 6, for the L02 cell hyperglycemic model, the cell survival rate of all examples was higher than that of the model group, while 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 cocrystal composition has a good protective effect on L02 hyperglycemic cells, and the effect of the glycoside cocrystal and the polypeptide composition is better.

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

[0083]

[0084] As shown in Table 7, for the L02 cell inflammation model, the cell survival rate of all examples was higher than that of the model group, while the IL-6 and TNFα levels were lower than those of the model group. Example 7 showed the highest cell survival rate and the lowest IL-6 and TNFα levels, indicating that the glycoside cocrystal composition has a good protective effect on L02 inflammatory cells, and the glycoside cocrystal and peptide composition are even more effective.

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

[0086]

[0087] As shown in Table 8, for the mouse MAFLD model: in terms of liver index, compared with the model group, the liver index of mice in all examples was significantly reduced, with the lowest liver index in Example 7; in terms of liver function, compared with the model group, the TBA, ALT, and AST levels of mice in all examples were significantly reduced, with the lowest TBA, ALT, and AST levels in Example 7; in terms of blood lipids, compared with the model group, all examples reduced the levels of TG and TG in mouse serum, with the lowest TC and TG levels in Example 7. This indicates that the glycoside cocrystal composition has a certain therapeutic effect on MAFLD in mice and a certain protective effect on liver function in mice. Furthermore, the effect of the glycoside cocrystal and the polypeptide composition is even better.

[0088] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that several improvements and modifications can be made without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered within the scope of the claims of the present invention.

Claims

1. A medicament for the prevention and / or treatment of metabolic-related fatty liver disease, characterized in that, The drug is a cocrystal of oleuropein-nicotinamide, cocrystal of luteolin-lysine, and a polypeptide with the amino acid sequence SEQ ID NO:1, in a mass ratio of 1:1:

1. The oleuropein-nicotinamide co-crystal is prepared by placing 0.1 mmol of oleuropein and 0.4 mmol of nicotinamide in a ball mill, adding 3 drops of anhydrous ethanol, grinding, and then drying the resulting powder under vacuum. The preparation method of the luteolin-lysine co-crystal includes 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 resulting powder was dried in a vacuum drying oven for 12 h to obtain luteolin-lysine cocrystal.

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