PHARMACEUTICAL COMPOSITION FOR PREVENTING OR TREATING NONALCOHOLIC STEATOHEPATITIS CONTAINING ASTER KORAIENSIS NAKAI EXTRACT

Abstract

A pharmaceutical composition containing an Aster koraiensis Nakai extract as an active ingredient, of the present invention, inhibits fat accumulation in liver tissue and treats lobular inflammation, and inhibits ballooning degeneration so as to prevent, alleviate or treat nonalcoholic steatohepatitis.

Claims

1. A pharmaceutical composition, containing a Gymnaster koraiensis extract as an active ingredient, for preventing or treating nonalcoholic steatohepatitis.

2. The pharmaceutical composition of claim 1, wherein the Gymnaster koraiensis extract is obtained by extraction with at least one solvent selected from the group consisting of water, a C1-C4 lower alcohol, acetone, ethyl acetate, and hexane.

3. The pharmaceutical composition of claim 2, wherein the C1-C4 lower alcohol is methanol or ethanol.

4. The pharmaceutical composition of claim 1, wherein the Gymnaster koraiensis extract is obtained by extraction from an aerial part including a flower of Gymnaster koraiensis or extraction from a flower of Gymnaster koraiensis.

5. A health functional food composition, containing a Gymnaster koraiensis extract as an active ingredient, for preventing or alleviating nonalcoholic steatohepatitis.

6. The health functional food composition of claim 5, wherein the Gymnaster koraiensis extract is obtained by extraction with at least one solvent selected from the group consisting of water, a C1-C4 lower alcohol, acetone, ethyl acetate, and hexane.

7. The health functional food composition of claim 6, wherein the C1-C4 lower alcohol is methanol or ethanol.

8. The health functional food composition of claim 5, wherein the Gymnaster koraiensis extract is obtained by extraction from an aerial part including a flower of Gymnaster koraiensis or extraction from a flower of Gymnaster koraiensis.

9. A method for preventing or treating nonalcoholic steatohepatitis, comprising administering to in a subject in need thereof an effective amount of a Gymnaster koraiensis extract.

10. The method of claim 9, wherein the Gymnaster koraiensis extract is obtained by extraction with at least one solvent selected from the group consisting of water, a C1-C4 lower alcohol, acetone, ethyl acetate, and hexane.

11. The method of claim 10, wherein the C1-C4 lower alcohol is methanol or ethanol.

12. The method of claim 9, wherein the Gymnaster koraiensis extract is obtained by extraction from an aerial part including a flower of Gymnaster koraiensis or extraction from a flower of Gymnaster koraiensis.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0037] FIG. 1 is a diagram showing nonalcoholic fatty liver disease (NAFLD) activity scores of a normal group (normal), a control group (control), Example 1 (MJAK) administration groups, an Example 2 (MJAK-F) administration group, an Example 3 (MJAK-L) administration group, an Example 4 (MJAK-S) administration group, and positive control groups (silymarin and pioglitazone) in a high-fat diet fed model.

[0038] FIG. 2 is a diagram showing steatosis assessment results of a normal group, a control group, Example 1 to Example 4 administration groups, and positive control groups in a high-fat diet fed model.

[0039] FIG. 3 is a diagram showing lobular inflammation assessment results of a normal group, a control group, Example 1 to Example 4 administration groups, and positive control groups in a high-fat diet fed model.

[0040] FIG. 4 is a diagram showing ballooning assessment results of a normal group, a control group, Example 1 to Example 4 administration groups, and positive control groups in a high-fat diet fed model.

[0041] FIG. 5 shows Oil-red O staining results of mouse liver tissues of a normal group, a control group, Example 1 administration groups, and a positive control group in a high-fat diet fed model.

[0042] FIG. 6 shows H&E staining results of mouse liver tissues of a normal group, a control group, Example 1 administration groups, and a positive control group in a high-fat diet fed model.

[0043] FIG. 7 presents diagrams showing AST and ALT levels of a normal group, a control group, Example 1 administration groups, and a positive control group in a high-fat diet fed model.

BEST MODE FOR CARRYING OUT THE INVENTION

[0044] Hereinafter, preferable exemplary embodiments of the present disclosure will be described in detail. However, the present disclosure is not limited to the exemplary embodiments described herein, and thus may be embodied into different forms. These exemplary embodiments are provided so that the present disclosure will be thorough and complete and will fully convey the scope of the disclosure to those skilled in the art.

TEST EXAMPLE 1

Preparation of Gymnaster koraiensis extract

[0045] 1. Preparation of Gymnaster koraiensis Extract of Example 1

[0046] The aerial part including flowers of Gymnaster koraiensis was prepared by collection in August 2018 in Asan-si, Chungcheongnam-do, Korea. Gymnaster koraiensis, which had been cleanly washed with water and then dried to have a moisture content of 9.2% or less, was finely cut, and then 20 L of 95% ethanol was added to 1 kg thereof, followed by reflux extraction at 70° C. for 4 hours, and thereafter, the extract was filtered. To the residue remaining after the filtration of the extract was again added 20 L of 95% ethanol, and reflux extraction at 70° C. for 4 hours was further repeated two times, thereby obtaining 60 L of a total extract. The 60 L of extract was concentrated under reduced pressure at 45-55° C. to obtain 168.4 g of a 95% ethanol extract, and this extract was called a Gymnaster koraiensis extract of Example 1 (or called “MJAK”).

[0047] 2. Preparation of Gymnaster koraiensis extracts of Examples 2 to 4

[0048] The aerial part including flowers of Gymnaster koraiensis collected as in Example 1 was separated into a flower part of Gymnaster koraiensis and a part of leaves, stems, and branches of Gymnaster koraiensis, which were the other part of the aerial part not including flowers of Gymnaster koraiensis. These parts were cleanly washed with water, dried to have a moisture content of 9.2% or less, and then finely cut, thereby preparing 1 kg for each part. Extraction was performed by the same method as in Example 1 to obtain 32.3 g of a flower extract of Gymnaster koraiensis, assigned as Example 2 (or “MJAK-F”), and 170.2 g of an extract of leaves, stems, and branches of Gymnaster koraiensis, assigned as Example 3 (or “MJAK-L”).

[0049] In addition, young sprouts collected in April 2018 in Asan-si, Chungcheongnam-do, Korea, were cleanly washed with water, dried to have a moisture content of 9.2% or less, and then finely cut, thereby preparing 1 kg. Extraction was performed by the same method as in Example 1 to obtain 121.6 g of a young sprout extract of Gymnaster koraiensis, assigned as Example 4 (or “MJAK-S”).

TEST EXAMPLE 2

Efficacy Assessment in High-Fat Diet (HFD) Fed Nonalcoholic Steatohepatitis Model

[0050] 1. Test Method

[0051] For the construction of a nonalcoholic fatty liver animal model, 5-week-old male C57/BL6J mice (Charles River Laboratories Japan Inc., Japan) were acclimatized for one week in an animal test facility where a temperature of 22-25° C., a relative humidity of 50-60%, a light-dark cycle of 12 hours, and an illuminance of 200-300 LUX were maintained. Two mice were housed in each polycarbonate box for mice with regular feed (Daehan Biolink Co., Ltd., Republic of Korea) and drinking water ad libitum during the acclimatization period of one week.

[0052] After the acclimatization, eight animals per group were grouped as follows such that the average body weights were similar. A normal group was fed a normal diet (Rodent Diet with 10% kcal fat, Republic of Korea), and a control group, groups administered Gymnaster koraiensis extracts of Examples 1 to 4, and a silymarin or pioglitazone administration group as a positive control group, were fed a high-fat diet (60% kcal high fat diet, Research Diets Inc. NJ, USA) ad libitum for 12 weeks.

TABLE-US-00001 TABLE 1 Test group configuration and condition in high-fat diet fed nonalcoholic steatohepatitis model Test group Condition G1 Normal group Normal diet + 0.5% CMC G2 Control group High-fat diet + 0.5% CMC G3 Example 1 (MJAK) low High-fat diet + 0.5% CMC + MJAK 125 mg/kg G4 Example 1 (MJAK) medium High-fat diet + 0.5% CMC + MJAK 250 mg/kg G5 Example 1 (MJAK) high High-fat diet + 0.5% CMC + MJAK 500 mg/kg G6 Example 2 (MJAK-F) medium High-fat diet + 0.5% CMC + MJAK 250 mg/kg G7 Example 3 (MJAK-L) medium High-fat diet + 0.5% CMC + MJAK 250 mg/kg G8 Example 4 (MJAK-S) medium High-fat diet + 0.5% CMC + MJAK 250 mg/kg G9 Positive control Silymarin High-fat diet + 0.5% CMC + silymarin 200 mg/kg G10 Pioglitazone High-fat diet + 0.5% CMC + pioglitazone 10 mg/kg

[0053] The doses of silymarin and pioglitazone as positive control groups were set to 200 mg/kg and 10 mg/kg, respectively, with reference to the reported toxicity and side effects thereof. The normal group was orally administered a 0.5% carboxymethylcellulose (CMC) solution once a day in the morning for 84 days, a total of 84 times, from the day of feeding of the normal diet. The control group was orally administered a 0.5% CMC solution once a day in the morning for 84 days, a total of 84 times, from the day of feeding of the high-fat diet. The Examples 1 to 4 administration groups and the positive control groups were orally administered Example 1 (125, 250, and 500 mg/kg), Examples 2 to 4 (250 mg/kg), silymarin 200 mg/kg, and pioglitazone 10 mg/kg, respectively, which were added to a 0.5% CMC solution, once a day in the morning for 84 days, a total of 84 times, from the day of feeding of the high-fat diet.

[0054] 2. Histopathological Assessment Methods and Results

[0055] (1) Preparation of H&E Staining Slides and Oil-Red O Staining Slides

[0056] For histopathological examination, the mice were sacrificed and the liver tissue was resected to prepare hematoxylin-eosin (H&E) stained slides and Oil-red O staining slides. H&E staining is a staining method that can broadly identify the whole sample and is performed to observe a change in cell size due to adipogenesis in the liver tissue and fat globules generated among cells. Oil-red O staining is performed to determine the amount of fat produced in the liver tissue.

[0057] The resected liver tissue of mice was fixed in a 4% paraformaldehyde solution at 4° C. for 3 days, transferred into a 30% sucrose solution, and stored at 4° C. before Oil-red O staining. The tissue for hematoxylin & eosin (H&E) staining was immersed and fixed in 10% formalin.

[0058] First, the tissue for Oil-red-0 staining was cryo-sectioned into a thickness of 10 μm, attached to tissue slides, rehydrated, subjected to Oil-red-O staining, dehydrated, and then sealed.

[0059] The tissue for H&E staining fixed in 10% formalin was sectioned into a thickness of 5 μm, attached to tissue slides, stained with a hematoxylin solution and an eosin solution in that order, dehydrated, and then sealed.

[0060] (2) Histopathological Assessment Results

[0061] The H&E-stained slides were histopathologically assessed under a microscope, and the nonalcoholic fatty liver disease (NAFLD) activity score (NAS) based on the criteria on Table 2 was numerically recorded. The nonalcoholic fatty liver disease activity score is expressed as a sum of assessment scores for changes in 1) steatosis, 2) lesions such as lobular inflammation, and 3) hepatocellular ballooning, and is widely used for evaluating the severity of nonalcoholic steatohepatitis.

TABLE-US-00002 TABLE 2 NAFLD activity score (NAS) assessment criteria Item Definition Score Steatosis Low- to medium-power evaluation of parenchymal involvement by steatosis <5% 0 5~33% 1 >33~66% 2 >66% 3 Lobular Overall assessment of all inflammatory foci Inflammation No foci 0 <2 foci per 200x field 1 2-4 foci per 200x field 2 >4 foci per 200x field 3 Ballooning None 0 Few balloon cells 1 Many cells/prominent ballooning 2

[0062] As shown in FIG. 1, the groups administered the Gymnaster koraiensis extract (MJAK) of Example 1 were identified to show a significant decrease in NAFLD activity score in a dose-dependent manner compared with the control group or the silymarin or pioglitazone administration group. The Example 1 (500 mg/kg) administration group was identified to have a NAFLD activity score equal to or lower than that of the normal group.

[0063] As shown in FIG. 2, the groups administered the Gymnaster koraiensis extract of Example 1 showed a significantly excellent effect of inhibiting steatosis in the liver tissue compared with the control group, the silymarin group, and the pioglitazone group. Furthermore, the Example 1 administration groups were identified to have excellent effects of mitigating lobular inflammatory cell infiltration and inhibiting ballooning in a dose-dependent manner compared with the silymarin or pioglitazone administration group (FIGS. 3 and 4).

[0064] As for the effects of the extracts of different parts of Gymnaster koraiensis, the groups administered the extract of the aerial part including flowers of Example 1 (MJAK), the extract of flowers of Example 2 (MJAK-F), the extract of leaves, stems, and branches of Example 3 (MJAK-L), and the extract of young sprouts of Example 4 (MJAK-S) at 250 mg/kg were identified to show a decrease in NAFLD activity score and have effects of inhibiting steatosis in the liver tissue, lobular inflammation, and ballooning, compared with the control group. In particular, the extract of the aerial part including flowers of Gymnaster koraiensis of Example 1 or the extract of flowers of Gymnaster koraiensis of Example 2 was identified to show a significant decrease in NAFLD activity score and be at three times excellent in terms of the effects of inhibiting steatosis in the liver tissue, ballooning, and lobular inflammation, compared with the extract of leaves, stems, and branches of Gymnaster koraiensis of Example 3 or the extract of young sprouts of Gymnaster koraiensis of Example 4 (see FIGS. 1 to 4).

[0065] FIG. 5 shows the results of observing the Oil-red O stained slides. It was identified that large fat globules were distributed throughout the liver tissue of the control group, but it was observed that the size of fat globules was decreased in a concentration-dependent manner by administration of Example 1 at different concentrations, and the liver tissue by the example at 500 mg/kg was the same as that of the normal group.

[0066] FIG. 6 shows the results of observing the H&E stained slides. The control group was observed to have an increase in size of liver tissue cells and adipogenesis thereamong, but it was identified that cell hypertrophy disappeared and the amount of fat produced among cells was significantly decreased, by administration of Example 1 at different concentrations.

[0067] It was therefore identified that the present disclosure was effective in the prevention or treatment of nonalcoholic steatohepatitis.

[0068] 3. Clinicopathological Assessment Results

[0069] (1) AST and ALT Analysis Methods

[0070] After completion of the high-fat diet feeding test for C57/BL6J mice, the mice were fasted for 12 hours before sacrifice, and then blood was collected from the abdominal aorta of C57/BL6J mice, followed by centrifugation to separate serum, which was then used for aspartate aminotransferase (AST) and alanine aminotransferase (ALT) analyses.

[0071] (2) AST and ALT Analysis Results

[0072] AST and ALT in FIG. 7 are blood indicators that indicating liver functions and, especially, used as indicators of liver cytotoxicity. The control group induced with nonalcoholic fatty liver showed an increase in AST by about 1.7 times and an increase in ALT by about 6.7 times compared with the normal group, whereas the increases in AST and ALT levels were significantly suppressed in a concentration-dependent manner by the administration of Example 1 (MJAK) at different concentrations.

[0073] It was identified through the efficacy assessments in the nonalcoholic steatohepatitis animal model that the Gymnaster koraiensis extracts had excellent effects on the prevention, alleviation, and treatment of nonalcoholic steatohepatitis.