KR102628223B1 - Pharmaceutical composition for preventing or treating liver damage comprising norgalanthamine - Google Patents

Abstract

It relates to a composition that can be used to prevent and treat liver damage and improve liver function, containing norgalantamine as an active ingredient. Norgalantamine increases serum alanine aminotransferase, aspartate aminotransferase, and total bilirubin in the damaged liver. Significantly reverses the increase and decrease in serum glucose levels, restores the activity of antioxidant enzymes, reduces the expression of genes involved in lipid accumulation and lipid synthesis, and improves inflammation and fibrosis, so it is used to prevent or treat liver damage. It can be usefully used.

Description

Composition for preventing or treating liver damage comprising norgalantamine {PHARMACEUTICAL COMPOSITION FOR PREVENTING OR TREATING LIVER DAMAGE COMPRISING NORGALANTHAMINE}

The present invention relates to a composition containing norgalantamine as an active ingredient that can be used to prevent and treat liver damage and improve liver function.

The liver is one of the important organs of the human body that is responsible for the body's metabolic processes, appropriately converting ingested food into the form of nutrients needed by various tissues and disposing of waste products remaining after use in the tissues. Specifically, it secretes bile, a digestive juice, metabolizes proteins, carbohydrates, and fats, stores glycogen and fat-soluble vitamins, synthesizes blood coagulation factors, removes waste and toxic substances from the blood, regulates blood volume, and prevents aging. It performs functions such as destroying red blood cells.

A variety of factors can cause liver damage, including mental stress, excessive consumption of fatty foods or alcohol, viral infection, exposure to harmful substances such as drugs or smoking, pollutants, and nutritional deficiencies. However, because the liver is an organ with a large buffering capacity, symptoms do not appear in the early stages of damage, and because there are few nerves that sense pain, liver damage is usually discovered only when it has significantly worsened into a disease such as cirrhosis. Cirrhosis and liver cancer are common final stages when various liver diseases progress chronically, and the liver is an organ for which early health management is very important.

Accordingly, studies have been conducted to provide compositions for treating liver damage using natural substances that can be safely used. For example, Patent Document 1 discloses a pharmaceutical composition for preventing or treating liver disease containing a fraction of ginseng leaf extract. .

1. Republic of Korea Patent No. 10-2204912

The purpose of the present invention is to provide a pharmaceutical composition with excellent effects in preventing or treating liver damage, and an anti-obesity composition with excellent effects on inhibiting fat accumulation. As a result of research to discover substances useful for liver damage, the present inventors found that norgalantamine It significantly reverses the increase in serum alanine aminotransferase, aspartate aminotransferase and total bilirubin levels and the decrease in serum glucose levels in the damaged liver, restores the activity of antioxidant enzymes, and promotes lipid accumulation and lipid synthesis. The present invention was completed by confirming that gene expression was reduced and inflammation and fibrosis were improved.

In order to achieve the above object, one aspect of the present invention provides a pharmaceutical composition for preventing or treating liver damage containing norgalantamine or a pharmaceutically acceptable salt thereof represented by the following formula (I) as an active ingredient.

[Formula I]

In the present invention, the compound of Formula 1 includes norgalantamine represented by Formula 1, its pharmaceutically acceptable salt, as well as its hydrate, solvate, and stereoisomer.

Norgalantamine, also called N-Desmethyl Galanthamine, is known to have anti-hair loss and hair growth-promoting effects after being isolated from the extract of Munjuran, but its liver protection effect is not yet known.

As used herein, the term “liver damage” refers to a condition in which liver cells or liver tissue cannot perform normal functions due to hepatitis virus, stress, alcohol, drugs, compounds, or excessive fat intake, including fatty liver, hepatitis, liver Includes conditions in which liver diseases such as fibrosis and cirrhosis have occurred.

According to one embodiment of the present invention, the norgalantamine increases bilirubin, alanine aminotransferase (ALT), and aspartate aminotransferase in a liver injury model induced by carbon tetrachloride (CCl ₄ ). It can reduce hepatocyte damage factors selected from the group consisting of aminotransferase (AST) (Figure 2).

Among the above factors, ALT and AST are present in the liver in amounts 7,000 and 3,000 times higher than in serum, respectively. When liver damage occurs, they leak into the serum and become an indicator of liver disease depending on the degree of elevation of the level in the serum. Bilirubin is a product of hemoglobin metabolism and is taken into the liver cells and excreted through the bile duct after a series of processes. When liver damage occurs, serum bilirubin increases.

Additionally, according to one embodiment of the present invention, norgalantamine can inhibit fibrosis, fat accumulation, or inflammation in hepatocytes or liver tissue.

Specifically, norgalantamine reduces blood glucose concentration increased by CCl ₄ ) administration (Figure 2D) and restores the activities of antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) (Figure 6). Since it has the effect of suppressing the expression of CYP1A2 and CYP2E1 genes, which are genes causing oxidative stress (Figure 5), liver damage can be suppressed.

In addition, norgalantamine inhibits the expression of genes involved in lipid accumulation and fat synthesis (PPAR-γ and aP2) (Figures 4 and 9), and inhibits the expression of pro-inflammatory mediators TNF-α, IL-1β, and MCP-1. Since the expression is down-regulated (Figure 7) and the Nrf2/HO-1 pathway is up-regulated (Figure 10), inflammation can be improved. Additionally, norgalantamine can reduce collagen deposition in liver tissue by downregulating the expression of fibrosis-related genes αSMA and fibronectin (Figures 11 and 12).

Norgalantamine of the present invention may be included in the composition in the form of a pharmaceutically acceptable salt. As the salt, an acid addition salt formed with a pharmaceutically acceptable free acid is useful. Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, as well as aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates and alkanes. Obtained from non-toxic organic acids such as dioates, aromatic acids, aliphatic and aromatic sulfonic acids. These pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, and iodine. Ide, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate , sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitro benzoate, hydroxybenzoate, methylbenzoate Toxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycol It may include nitrate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate or mandelate.

In addition, the acid addition salt can be prepared by a conventional method, for example, by dissolving the norgalanthamine in an excessive amount of aqueous acid and precipitating the salt with a water-miscible organic solvent, such as methanol, ethanol, acetone or acetonitrile. It can be manufactured.

Additionally, norgalantamine of the present invention can be used in the form of a pharmaceutically acceptable metal salt using a base. The alkali metal or alkaline earth metal salt can be obtained, for example, by dissolving the compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically appropriate to prepare sodium, potassium or calcium salts as metal salts. Additionally, the corresponding silver salt can be obtained by reacting an alkali metal or alkaline earth metal salt with an appropriate silver salt (eg, silver nitrate).

Additionally, the pharmaceutical composition according to one embodiment of the present invention may include a pharmaceutically acceptable carrier. The composition containing a pharmaceutically acceptable carrier may be in various oral or parenteral dosage forms.

When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations contain one or more compounds and at least one excipient, such as starch, calcium carbonate, sucrose, or lactose ( It is prepared by mixing lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is. Preparations for parenteral administration may include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable esters such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurel, glycerogelatin, etc. can be used.

In addition, the pharmaceutical composition is selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, oral solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. It can have any one formulation.

Additionally, the pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount. In the present invention, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the degree of liver damage, cause of liver damage, age, and gender. , can be determined based on factors including the activity of the drug, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used simultaneously, and other factors well known in the field of medicine. However, for desirable effects, the pharmaceutical composition of the present invention is preferably administered at 0.0001 to 100 mg/kg per day, preferably at 0.001 to 100 mg/kg. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And it can be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and can be easily determined by a person skilled in the art.

Another aspect of the present invention provides a food composition for preventing or improving liver damage containing norgalantamine or a pharmaceutically acceptable salt thereof as an active ingredient.

In the present invention, the compound of Formula 1 includes norgalantamine represented by Formula 1, its pharmaceutically acceptable salt, as well as its hydrate, solvate, and stereoisomer.

As described above, norgalantamine is excellent for improving liver damage, so it can be usefully used as a food composition for preventing or improving liver damage.

In the present invention, the food composition may be provided in the form of powder, granules, tablets, capsules, syrup, beverages, or pills, and is used with other foods or food additives in addition to the active ingredient norgalantamine, and is administered appropriately according to conventional methods. It can be used effectively. The mixing amount of the active ingredient can be appropriately determined depending on its purpose of use, for example, prevention, health, or therapeutic treatment.

The effective dose of the active ingredient contained in the food composition can be used in accordance with the effective dose of the pharmaceutical composition, but in the case of long-term intake for the purpose of health and hygiene or health control, it may be below the above range, Since the active ingredient poses no safety issues, it is certain that it can be used in amounts exceeding the above range.

The food composition includes ingredients commonly added during food production, for example, proteins, carbohydrates, fats, nutrients, seasonings, and flavoring agents. Examples of the above-mentioned carbohydrates include monosaccharides such as glucose, fructose, etc.; Disaccharides such as maltose, sucrose, oligosaccharides, etc.; and polysaccharides, such as common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As a flavoring agent, natural flavoring agents and synthetic flavoring agents can be used. For example, when the food composition of the present invention is manufactured as a drink, citric acid, high fructose corn syrup, sugar, glucose, acetic acid, malic acid, fruit juice, etc. may be additionally included in addition to the active ingredients of the present invention.

Meanwhile, the present inventors confirmed whether norgalantamine could suppress fat accumulation in normal adipocytes in addition to damaged liver tissue, and confirmed that the expression of genes related to fat accumulation and lipid synthesis were effectively suppressed (FIGS. 14 and 15) .

Accordingly, another aspect of the present invention provides an anti-obesity composition containing norgalantamine or a pharmaceutically acceptable salt thereof as an active ingredient.

As used herein, the term “anti-obesity” refers to improving symptoms of obesity, and obesity refers to a condition in which fat cells proliferate and differentiate in the body due to metabolic disorders, resulting in excessive accumulation of body fat.

As used herein, the term “improvement” refers to any action that improves or benefits the symptoms of an individual suspected of or suffering from obesity using the composition, and the anti-obesity composition includes PPAR-γ and aP2, which are involved in lipid synthesis. And the effect of suppressing the expression of SREBP-1c gene is significantly excellent.

The anti-obesity composition of the present invention may be used in the form of a pharmaceutical composition and/or a food composition, and may contain additional ingredients as described in the pharmaceutical composition for preventing or treating liver damage or the food composition for preventing or improving liver damage. It may be included or developed into a specific formulation.

Norgalantamine of the present invention significantly reverses the increase in serum alanine aminotransferase, aspartate aminotransferase and total bilirubin levels and the decrease in serum glucose level in the damaged liver, restores the activity of antioxidant enzymes, and reduces lipid accumulation. And it reduces the expression of genes involved in lipid synthesis and improves inflammation and fibrosis, so it can be usefully used to prevent or treat liver damage.

Figure 1 shows a method for inducing acute and chronic liver damage in mice and a norgalantamine administration schedule according to an example of the present invention.
Figure 2 shows the levels of ALT (A), AST (B), T-bil (C), and glucose (D) in the serum of liver-damaged mice administered norgalantamine: Normal - normal control group; Liver injury induction group administered only Vehicle-CCl4; NG1-CCl ₄ + norgalantamine 1 mg/kg administration group; NG10-CCl ₄ + norgalantamine 10 mg/kg administration group; and Sily100-CCl ₄ + Silymarin 100 mg/kg administration group.
Figure 3 shows the extent of damage improvement by norgalantamine administration in liver tissue of liver-damaged mice confirmed by tissue staining (A), damage grade assessment (B), and necrosis area (C).
Figure 4 shows changes in fat accumulation caused by norgalantamine administration in the liver tissue of liver-damaged mice using Oil Red O staining.
Figure 5 shows changes in mRNA expression of CYP1A2 and CYP2E1 due to norgalantamine administration in liver tissue of liver-damaged mice.
Figure 6 shows the antioxidant effect of norgalantamine in liver tissue of liver-damaged mice confirmed by the activities of SOD (Superoxide Dismutase) and catalase.
Figure 7 shows the mRNA levels of TNF-α, IL-1β, and MCP-1 in liver tissue of liver-damaged mice to confirm the anti-inflammatory effect of norgalantamine administration.
Figure 8 shows changes in Kupffer cell activation and inflammatory cell infiltration caused by norgalantamine administration in liver tissue of liver-damaged mice.
Figure 9 confirms changes in the expression of PPAR-γ (proliferator-activated receptor-γ) and aP2 (adipocyte fatty acid-binding protein-2) genes due to norgalantamine administration in damaged liver tissue.
Figure 10 shows changes in nuclear translocation of Nrf-2 (nuclear factor erythroid 2-related factor 2) (A) and changes in expression of HO-1 (heme oxygenase 1) (B) caused by norgalantamine administration in damaged liver tissue. will be.
Figure 11 confirms the hepatoprotective effect of norgalantamine administration in the liver tissue of mice with chronic liver damage.
Figure 12 confirms changes in the expression of αSMA (α-smooth muscle actin) and fibronectin mRNA due to norgalantamine administration in liver tissue of mice with chronic liver damage.
Figure 13 schematically shows the expected mechanism of norgalantamine to protect against liver damage and inhibit fat accumulation.
Figure 14 shows the degree of fat accumulation confirmed by Oil Red O staining after treating adipocytes with MDI (IBMX, dexa, insulin) and norgalantamine.
Figure 15 shows changes in expression of lipid synthesis genes PPAR-γ, aP2, and SREBP-1c (sterol regulatory element-binding protein-1c) genes after treating adipocytes with MDI (IBMX, dexa, insulin) and norgalantamine. has been confirmed.

Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are intended to illustrate one or more embodiments and the scope of the present invention is not limited to these examples.

Experimental method

1. Compounds and reagents

Norgalanthamine (D292035) was purchased from Toronto Research Chemicals (Canada), and silymarin, used as a positive control, was purchased from Sigma-Aldrich (USA). Colorimetric assay kits for measuring superoxide dismutase (SOD) and catalase (CAT) activities were purchased from Abcam (UK). Polyclonal antibodies targeting Nrf-2 (nuclear factor erythroid 2-related factor 2) and HO-1 (heme oxygenase 1) are from Cell Signaling Technology (USA), and Iba-1 (ionized calcium binding adapter molecule 1) is from Wako. Pure Chemical Industries, Ltd. Purchased from (Japan). ABC Elite kit and diaminobenzidine (DAB) substrate were purchased from Vector Laboratories (USA).

2. Experimental animals

Male C57BL/6 mice (DBL, Korea) weighing 20 to 22 g and 6 weeks old were used in all experiments. Mice were raised on a 12-hour light/dark cycle at a temperature of 25-28°C, fed standard food, and provided with water ad libitum.

3. Experimental design

For the acute model, mice were randomly divided into 5 groups of 6, and the norgalantamine-treated CCl ₄ -injured group was administered norgalantamine (1 or 10 mg/ml, respectively (NG1 and NG10), phosphate-buffered saline (PBS) for 7 consecutive days. ) was diluted to pH 7.4 and administered orally. In both acute and chronic models, PBS was used as a vehicle control and silymarin was used as a positive control (100 mg/kg; (Ni and Wang, 2016)), and both were administered according to the same protocol. Liver injury was induced by injecting a 1:1 (v/v) mixture of CCl ₄ and sterilized olive oil (ip, 1.5 mL/kg) into the abdominal cavity of mice. CCl ₄ was administered once 24 hours after the last administration of norgalantamine.

In the chronic model, mice were randomly divided into four groups of six mice each. The vehicle control group was administered PBS (pH 7.4), and the norgalantamine-treated CCl ₄ -damaged group was orally administered norgalantamine (10 mg/ml (NG10), diluted in PBS, pH 7.4) for 14 consecutive days. Liver injury was induced by injecting a 1:1 (v/v) mixture of CCl ₄ and sterilized olive oil (ip, 1.5 mL/kg) into the abdominal cavity of mice. CCl ₄ was injected every 72 hours for 14 days (Figure 1).

4. Sample collection and blood analysis

Mice were fasted after the final treatment and sacrificed 24 hours after induction of CCl ₄ damage in the acute model or administration of the last dose of CCl ₄ in the chronic model. After anesthetizing the mouse with isoflurane inhalation (Hana Pharm Co., Ltd., Seoul), blood was collected from the inferior vena cava, and the liver was dissected for histopathology, gene expression, and immunochemistry studies (Figure 1). Liver pieces were fixed in 10% neutral buffered formalin for histopathology or immediately frozen for RNA extraction. Blood samples were centrifuged at 13,000 rpm for 10 minutes at 4°C to separate serum. Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (T-bil), and glucose were measured with a Beckman Coulter AU680 analyzer (Beckman Coulter, Japan) according to the manufacturer's instructions.

5. Histopathological examination of liver damage

Paraffin was removed from paraffin-embedded liver sections (4 ㎛), and stained with hematoxylin and eosin (H&E) for light microscopy. The degree of necrosis after acute liver injury was assessed using an injury score based on the extent of necrotic lesions in the liver parenchyma (Table 1), with each sample scored independently by three pathologists blinded to each experimental group. It has been done. The scoring system is as follows: grade 0, no pathological changes; Grade 1, degenerative hepatocytes with rare foci of necrosis; Grade 2, small, mild central lobar necrosis around the central vein; Grade 3, mild central lobe necrosis more severe than grade 2; Grade 4, central lobe necrosis more severe than grade 3 (Dai et al., 2018; Li et al., 2013).

Areas of necrosis were assessed on paraffin-embedded sections stained with toluidine blue, and the severity of fatty changes was assessed on fixed frozen sections stained with Oil Red O, which detects neutral triglycerides and lipids. Picrosirius red (Polysciences, USA) staining was used for fibrillary collagen. Areas of lipid (area stained red as a percentage of the total area of the liver cross-section), necrosis (area stained light blue as a percentage of the total area of the liver cross-section), and collagen deposition (area stained in light blue as a percentage of the total area of the liver cross-section) The percentage of area stained in red) was evaluated with Aperio eSlide Manager software (Leica Biosystems, USA).

6. Antioxidant enzyme activity analysis

Mouse liver pieces were immediately frozen until use. The tissue was then homogenized in a pestle homogenizer, and SOD and CAT activities were measured using a commercial assay kit (Abcam) according to the manufacturer's protocol.

7. Western blot analysis

Cytoplasmic and nuclear fractions were separated with NE-PER® nuclear and cytoplasmic extraction reagent as recommended by the manufacturer (Thermo Scientific, USA). Proteins (40 μg/sample) were separated by 10% (w/v) sodium dodecyl sulfate or sodium lauryl sulfate polyacrylamide gel electrophoresis (SDS- and SLS-PAGE, respectively) and plated on nitrocellulose membranes (Schleicher and Schuell, Transferred to the United States).

Target proteins were immunodetected by reacting the membrane with the corresponding primary antibody (Table 2) for 2 hours. Proteins bound to antibodies were detected using a chemiluminescent substrate (Miracle-Star; iNtRON Biotech, Korea). The protein blot was then imaged and the density of the bands was analyzed using ImageJ software (NIH, Bethesda, MD, USA). β-Actin was used as an internal control.

The optical density (OD; per mm) of each band was measured and the density ratio for the β-actin band was determined with ImageJ. Data are expressed as mean ± standard error of the mean (SEM).

8. Quantitative real-time PCR

Gene expression levels were analyzed in mRNA extracted from mouse liver using TRIzol® reagent (Ambion, USA), CellScript ^TM all-in-one 5x first-strand cDNA, and cDNA Synthesis Master Mix (CellSafe, Korea). Real-time PCR was performed with Luna® Universal qPCR Master Mix (New England BioLabs, Ipswich, MA, USA). mRNA expression levels were calculated according to the 2-ΔΔCt method using GAPDH as an internal control. Primers used in PCR are listed in Table 3.

9. Immunohistochemistry

Liver sections were reacted with rabbit anti-Iba-1 antibody, and the peroxidase reaction was visualized with a DAB kit (Vector Laboratories, USA). Liver section slides were counterstained with hematoxylin and then mounted, and the area immunostained with Iba-1 antibody was semi-quantitatively analyzed using ImageJ (n=6 per group). Five different sections from each liver were evaluated and the positive area was calculated as a percentage of the total area of each section (n=5 per group).

10. Measurement of liver hydroxyproline content

Liver hydroxyproline content was quantified colorimetrically in flash-frozen liver samples using the hydroxyproline kit (Abcam) according to the manufacturer's protocol. The experimental results were quantified by comparison with a standard curve of known hydroxyproline concentration and expressed as mg hydroxyproline/mg liver.

11. Fat accumulation inhibition effect

3T3-L1 preadipocytes were cultured on plates and then treated with MDI (IBMX, dexa, insulin) to induce fat accumulation. The experimental group was treated with norgalantamine at concentrations of 5, 10, 20, and 40 μg/ml, respectively, and the positive control group was treated with silymarin at concentrations of 5, 10, and 20 μg/ml, respectively. After culturing for an additional 6 days, the cells were stained with Oil Red O, and changes in expression of genes related to lipid synthesis were confirmed.

Additionally, norgalantamine (0, 1, and 10 mg/kg) or silymarin (100 mg/kg) was administered to the chronic liver injury model for 14 days, and the mice were sacrificed on the 15th day.

12. Statistical analysis

All results were expressed as mean ± SEM. Statistical significance was defined as a P value <0.05 based on the results of one-way analysis of variance followed by Bonferroni's multiple comparison post-hoc test.

Experiment result

1. Check biochemical parameters in serum

The protective efficacy of norgalantamine against CCl ₄ -induced liver injury was evaluated by measuring serum levels of ALT, AST, and T-bil enzymes (Figure 2, A to C). Serum ALT, AST, and T-bil levels were in the normal range (31.8±2.20, 42.2±2.29, and 0.17±0.00 U/L, respectively) in the control group (Normal), whereas in the liver injury induction group (administration of only CCl ₄ ; Vehicle) were significantly higher (6,088.0±697.47 U/L, p<0.001; 4134.0±392.95, p<0.001; and 0.27±0.02, p<0.001, respectively).

However, in the norgalantamine administration group (1 mg/kg; NG 1), ALT was 4,176.0±441.97 U/L (p<0.05) and AST was 2,952.0±358.07 U/L (p<0.05), showing that the levels of both enzymes were significantly higher. decreased. Additionally, in the norgalantamine administration group (10 mg/kg; NG 10), ALT was 3,588.00±281.13 U/L (p 0.01), AST was 2,236.00±158.83 U/L (p<0.001), and T-bil was 0.22±0.01. As measured by U/L (p<0.05), a significant dose-dependent decrease was observed compared to the liver damage induction group (CCl ₄ only administered; Vehicle).

In the silymarin treatment group (positive control, 100 mg/kg; Sily 100), ALT was 3,588.00±225.73 U/L (p<0.01), AST was 1,840.00±185.66 U/L (p<0.001), and T-bil was 0.21. ±0.01 U/L (p<0.05), showing a significant decrease in enzyme levels compared to the liver damage induction group (administration of only CCl ₄ ; Vehicle).

The serum glucose level was 32.80±3.53 U/L in the liver injury induction group (CCl ₄ only administered; Vehicle), which was significantly lower than the normal control group's 296.60±17.87 U/L (p<0.001), but in the norgalantamine administered group ( In 10 mg/kg; NG 10), it was found to be 104.40±6.95 U/L, which was significantly higher than the liver damage induction group (administration of only CCl ₄ ; Vehicle).

2. Liver damage improvement effect of norgalantamine

As a result of histopathological study, it was confirmed that norgalantamine improved CCl ₄ -induced liver damage (Figure 3). The liver of the Normal Control group showed normal tissue structure including hepatocytes with well-preserved cytoplasm, prominent nuclei, and central veins (Figure 3A). However, in the liver of the liver injury induction group (Vehicle), loss of liver structure, inflammatory cell infiltration, and cell edema along with a large area of pericentral necrosis were observed (Figure 3B). Liver lesions appeared to be more improved in the norgalantamine-treated group (NG1 and NG10) (Figure 3C and D) than in the silymarin-treated group (Sily100) (Figure 3E).

Liver injury grade was assessed based on H&E staining. According to the histological score, the norgalantamine administration group (NG1 and NG10) significantly suppressed acute liver damage compared to the liver injury induction group (Vehicle) (p<0.001). In the silymarin-administered group (Sily100), a liver damage inhibition effect similar to that of norgalantamine was observed (Figure 3F).

As a result of evaluating the degree of central lobe necrosis around the central vein using toluidine blue staining, the necrotic area that increased in the liver injury induction group (Vehicle) was significantly reduced in the norgalantamine-administered group (NG10) and the silymarin-administered group (Sily100) (Figure G of 3).

Additionally, fatty changes were examined in the liver tissue of mice (Figure 4). In the normal control group (Normal), no lipid droplets were detected by Oil Red O staining (Figure 4A), whereas in the liver damage-induced group (Vehicle), diffuse fatty degeneration was observed throughout the liver (Figure 4B). In contrast, the degree of fatty change in the liver was relatively reduced in the norgalantamine-administered group (NG1 and NG10) and the silymarin-administered group (Sily100 (Figure 4C to E). The proportion of Oil-Red-O-positive area was compared to the normal control group ( It was significantly higher in the liver damage induction group (Vehicle) than in the normal cont. (p<0.001), but significantly higher in the norgalantamine administration group (NG1 and NG10) (p <0.001) and silymarin administration group (Sily100) (p <0.001). (Figure 3F). These results mean that norgalantamine treatment can alleviate CCl ₄ -induced histopathological changes in mouse liver.

3. Regulation of CYP1A2 and CYP2E1 gene expression by norgalantamine in damaged liver

The mRNA expression levels of CYP1A2 and CYP2E1 in the liver were analyzed by qPCR. CYP 2E1 mRNA expression was significantly decreased in all administration groups compared to the normal control group (liver damage induction group (Vehicle) and NG10, p<0.01; NG 1 and silymarin administration group (Sily100), p<0.05). However, there was no change in expression levels in all experimental groups (Figure 5A).

On the other hand, CYP1A2 mRNA expression was significantly decreased in the liver injury induced group (Vehicle) compared to the normal control group, and significantly increased in the NG10 (p<0.05) and silymarin administered group (Sily100) (p<0.001) (Figure 5B) ).

4. Antioxidant effect of norgalantamine on damaged liver

The antioxidant effect of norgalantamine on liver damage was confirmed by measuring the activities of SOD and CAT. Compared to the normal control group (Normal), SOD and CAT activities were significantly reduced in the liver injury induction group (Vehicle) (80.37±5.24 and 0.46±0.04 U/L, respectively; all p<0.05). However, in the norgalantamine administration group (NG1 and NG10), the activities of both enzymes were significantly improved compared to the liver damage induction group (Vehicle), and in particular, in NG10, SOD activity rose to a significantly high level (102.22 ± 2.26 U/L, p <0.01; Figure 6A). CAT activity was also significantly elevated in NG1 and NG10 (0.62±0.03, p<0.01; 0.98±0.21, p<0.05, respectively, Figure 6B). These results imply that the hepatoprotective effect of norgalantamine derives from its antioxidant activity.

5. Anti-inflammatory effects of norgalantamine on damaged liver

To confirm the anti-inflammatory effect of norgalantamine, TNF-α, IL-1β, and MCP-1 mRNA levels were measured in the liver of mice (Figure 7).

The mRNA expression of TNF-α and IL-1β was 3.41±0.56 and 2.37±0.27 U/L, respectively, in the liver injury-induced group (Vehicle), and 0.71±0.26 and 1.42±0.45 U/L, respectively, in the normal control group (Normal). In the liver damage induction group (Vehicle), it significantly increased (p<0.01), and in the norgalantamine administration group (NG1 and NG10), it significantly decreased to 2.30±0.27 and 1.47±0.02 U/L, respectively (all p<0.05). ; Figure 7A and B).

Additionally, the mRNA level of MCP-1, an inflammatory chemokine, was significantly higher in the liver injury-induced group (Vehicle) than the normal control group (Normal) (12.37±1.00 vs. 1.31±0.27 U/L, p <0.01), norgalantamine. In the administration groups (NG1 and NG10), liver damage was significantly lower than that in the induced group (Vehicle) (6.94 1.3-fold change and 6.9±0.77-fold change, respectively, both p<0.01; Figure 7C).

Kupffer cells/macrophages are activated during liver injury. In the normal control group (Normal), Iba-1-positive Kupffer cells were observed along the sinusoids (Figure 8A), but no infiltration of inflammatory cells was observed. In contrast, activated Kupffer cells and inflammatory cell infiltration were confirmed in the liver injury induction group (Vehicle) (Figure 8B). However, in the norgalantamine-treated groups (NG1 and NG10), the number of Iba-1-positive Kupffer cells decreased (Figure 8, D and E). Additionally, the proportion of Iba-1 positive areas was significantly higher in the liver injury induced group (Vehicle) compared to the normal control group (Normal) (p<0.01), but NG10 (p<0.01) and silymarin administered group (Sily100) (p<0.01) 0.01), it decreased (F in Figure 8).

6. Inhibitory effect of norgalantamine on fat accumulation in damaged liver

By analyzing the mRNA levels of PPAR-γ (proliferator-activated receptor-γ) and aP2 (adipocyte fatty acid-binding protein-2) genes involved in lipid absorption and metabolism in damaged liver tissue, the effect of norgalantamine on suppressing fat accumulation was examined. evaluated (Figure 9).

As a result, the expression levels of both genes were significantly increased in the liver damage induction group (Vehicle) (p<0.01), while the expression levels of both genes were significantly decreased in the norgalantamine administration group (NG1 and NG10) (p<0.05) ).

7. The effect of norgalantamine on improving Nrf-2 and HO-1 levels in damaged liver

Nrf-2 and its downstream target HO-1 play an important role in tissue protection by regulating the expression of cytoprotective and antioxidant genes together with other antioxidant enzymes (e.g. SOD and glutathione peroxidase). Western blotting was performed to determine the effect of norgalantamine on Nrf-2 levels in the cytoplasm and nucleus of hepatocytes. Compared to the liver damage-inducing group (Vehicle), the cytoplasmic level of Nrf-2 and the degree of nuclear translocation of the protein were significantly increased in the norgalantamine-administered group (NG1 and NG10) (Figure 10A). Consistent with these results, the HO-1 protein level was significantly higher in NG10 than in the liver damage induction group (Vehicle) (Figure 10B).

8. Norgalantamine’s protective effect against chronic liver damage

As a result of staining the liver section with Picrosirius Red, collagen fibers were confirmed around the peripheral venular region of the normal control group (Figure 11A). However, in the liver damage induction group (Vehicle), collagen tissue proliferation due to fibrosis was detected (Figure 11B). Compared to the liver damage-inducing group (Vehicle), the proliferation of collagen tissue in the liver was significantly reduced in the norgalantamine-administered group (NG1 and NG10) and the silymarin-administered group (Sily100) (FIG. 11C and D).

The proportion of Picrosirius Red positive area was significantly higher in the liver injury induced group (Vehicle) (3.77±0.27%, p<0.01) than in the normal control group (1.60±0.14%). Compared to the liver damage inducing group (Vehicle), the rate was significantly lower in the norgalantamine administered group (NG10) (3.00±0.02%, p<0.05) (Figure 11E).

Hydroxyproline levels in liver tissue were significantly increased in the liver injury induction group (Vehicle) (189.26±35.53 g, p<0.05) than in the normal control group (33.87±5.81 g), and in the norgalantamine administration group (NG10) (148.6±148.6±148.6 g, p<0.05). 24.94g, p<0.05) was significantly lower (Figure 11F).

Changes in biochemical serum and histopathological parameters in CCl ₄ -induced chronic liver injury mice were similar to those in CCl ₄ -induced acute liver injury mice (data not shown). In addition, αSMA and fibronectin mRNA changed 5.24 ± 1.75-fold and 1.48 ± 0.07 (all p < 0.05), respectively, in the liver injury-induced group (Vehicle), and 3.33 ± 1.76-fold each in the norgalantamine-administered group (NG10) (p <0.01) and a 1.03±0.15-fold change (p<0.05), which was significantly decreased in the norgalantamine administration group (NG10) (FIG. 12).

Based on the results so far, in mouse models of liver injury, norgalantamine suppresses oxidative stress, hepatic lipid accumulation, and inflammation through activation of antioxidant molecules, and expression of lipogenic markers, pro-inflammatory mediators, and fibrosis factors related to lipid accumulation. was confirmed to decrease (Figure 13). This shows that norgalantamine has excellent liver damage protection effects and fat accumulation inhibition effects.

9. Inhibitory effect of norgalantamine on fat accumulation

Meanwhile, it was confirmed whether norgalantamine could inhibit fat accumulation in normal adipocytes in addition to damaged liver tissue. As a result, norgalantamine significantly suppressed fat accumulation induced by MDI (Figure 14) and inhibited the expression of PPAR-γ, aP2, and SREBP-1c (sterol regulatory element-binding protein-1c) genes involved in fat synthesis. It was confirmed that expression was significantly suppressed (Figure 15).

Claims (7)

A pharmaceutical composition for preventing or treating liver damage caused by carbon tetrachloride, comprising norgalantamine represented by the following formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient.
[Formula I]
delete The pharmaceutical composition for preventing or treating liver damage caused by carbon tetrachloride according to claim 1, wherein the composition reduces hepatocyte damage factors selected from the group consisting of bilirubin, alanine aminotransferase, and aspartate aminotransferase. . The pharmaceutical composition for preventing or treating liver damage caused by carbon tetrachloride according to claim 1, wherein the composition inhibits fibrosis, fat accumulation, or inflammation of hepatocytes or liver tissue. A food composition for preventing or improving liver damage caused by carbon tetrachloride, comprising norgalantamine or a pharmaceutically acceptable salt thereof represented by the following formula (I) as an active ingredient.
[Formula I]
delete delete