KR102451161B1 - Composition for the Improvement and Protection of Liver Health Comprising Extract of Hibiscus Syriacus - Google Patents

Abstract

The present invention relates to a composition for improving and protecting liver health comprising an extract of Mugunghwa as an active ingredient.
Mugunghwa extract of the present invention does not show toxicity to the human body, has the effect of resolving oxidative stress, directly protecting hepatocytes, directly resolving the cytotoxicity of acetaldehyde, and effectively reducing the production of reactive oxygen species (ROS). It inhibits liver cell membrane damage, has antioxidant activity, and significantly reduces fat absorption in hepatocytes, thereby having an excellent effect on improving liver function and protecting the liver.
In addition, the composition of the present invention using a natural plant extract is not only very safe for the human body, but also has excellent stability, so that it can be usefully used for the purpose of improving liver function or protecting the liver in the fields of food and pharmaceuticals.

Description

Composition for the Improvement and Protection of Liver Health Comprising Extract of Hibiscus Syriacus comprising Mugunghwa extract as an active ingredient

The present invention relates to a composition for improving and protecting liver health, and more particularly, to a composition for improving and protecting liver health comprising an extract of Mugunghwa as an active ingredient.

The liver is one of the important organs of the human body that is responsible for the body's metabolic process that appropriately transforms the ingested food into the form of nutrients required by various tissues and processes the waste products remaining after being used by 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 wastes and toxic substances from the blood, regulates blood volume, and senescence. It performs functions such as destroying red blood cells.

Various factors, such as mental stress, excessive intake of food or alcohol containing fat, viral infection, drug or smoking, exposure to harmful substances such as pollutants, and malnutrition, can cause liver dysfunction. In addition, liver failure may cause other diseases by causing abnormalities in the immune system because the body does not have a protective and detoxifying action. Impaired liver function causes a variety of symptoms, such as weakness, low blood pressure, frequent strains and bleeding, tremors, and fluid accumulation in the abdominal cavity.

Various liver diseases can progress continuously to liver fibrosis and liver cancer if appropriate treatment is not performed. Accordingly, there are currently commercially available hepatoprotective agents such as UDCA (ursodeoxycholic acid) and silymarin, but their hepatocellular damage protection effect is also insufficient. high situation.

Accordingly, many natural products with hepatotoxic activity are being searched for and developed, but only a few cases are actually being used as therapeutic agents or undergoing clinical trials (Thabrew MI and Hughes RD., Phytother. Res., 10:461-467, 1996).

On the other hand, as a prior art for a composition for liver protection, Korean Patent Registration No. 10-1342489 (a composition for liver protection containing an extract of sagebrush) and Korean Patent Publication No. 10-2017-0036254 (a fermented product of shiitake mushroom for improving liver function) manufacturing method) and the like. In addition, in the development of liver function improving agents, drugs with liver effects, beverages, and health functional food materials, product development has been mainly focused on simple extracts of herbal medicines with proven liver function improvement effects. Mushroom mycelium extract, milk thistle extract (silymarin preparation), bokbunja extract, and lactic acid bacteria fermented kelp extract have been developed and used as individually recognized health functional food materials. However, many types of compositions related to liver function improvement and hangover relief have been commercialized and sold, but they do not achieve great effectiveness.

Therefore, it is necessary and urgent to develop a material capable of effectively treating liver disease along with liver protection by improving liver function.

Under this background, the present inventors conducted a study on a substance having excellent liver function improvement and hepatoprotective effect, and excellent liver function improvement and hepatoprotective effect, in particular, Mugunghwa extract among domestic native plants, exceeding the effect of commercially used drugs. By elucidating that it represents, the present invention was completed.

Accordingly, it is a main object of the present invention to provide a pharmaceutical composition for the treatment or prevention of liver disease comprising a Mugunghwa extract as an active ingredient.

Another object of the present invention is to provide a food composition for improving and protecting liver health comprising an extract of Mugunghwa as an active ingredient.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a pharmaceutical composition for the treatment or prevention of liver disease comprising an extract of Mugunghwa as an active ingredient.

The present inventors have conducted a study on a substance having excellent liver function improvement and liver protection effects, and among native plants in Korea, in particular, Mugunghwa extract does not show toxicity to the human body, H ₂ O ₂ and alcoholic oxidative stress are resolved, , has the effect of directly protecting hepatocytes, directly resolving the cytotoxicity of acetaldehyde, effectively inhibiting the production of reactive oxygen species (ROS), inhibiting liver cell membrane damage, and having antioxidant activity, and at the same time having antioxidant activity. It was confirmed that it was effective for improving liver function and protecting the liver by significantly reducing absorption, and it was confirmed that it can be used for the purpose of treating or preventing liver disease.

"Mugunghwa (Hibiscus syriacus)" of the present invention means a deciduous shrub of the mallow family, a dicotyledonous plant that grows wild throughout the Korean Peninsula. The Mugunghwa extract of the present invention may be extracted from natural, hybrid or mutated plants of Mugunghwa, and may also be extracted from plant tissue culture.

As used in the present invention, the term "extract" refers to an extract obtained by extraction of plants, a diluted or concentrated liquid of the extract, a dried product obtained by drying the extract, a prepared or purified product of the extract, or a mixture thereof, such as an extract It includes extracts of all formulations that can be formed by themselves and using extracts. The extract of the present invention may be prepared and used in the form of a dry powder after extraction.

In the extraction of the Mugunghwa of the present invention, the method of extracting the extract is not particularly limited, and may be extracted according to a method commonly used in the art. Non-limiting examples of the extraction method include hot water extraction, ultrasonic extraction, filtration, reflux extraction, and the like, and these may be performed alone or in combination of two or more extraction methods.

In the present invention, the type of extraction solvent used for extracting Mugunghwa is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the extraction solvent include water; C1-C4 lower alcohols, such as methanol, ethanol, propyl alcohol, and butyl alcohol; polyhydric alcohols such as glycerin, butylene glycol and propylene glycol; and hydrocarbon solvents such as methyl acetate, ethyl acetate, acetone, benzene, hexane, diethyl ether, and dichloromethane; Or a mixture thereof may be used, and preferably, water, lower alcohol, 1,3-butylene glycol, and ethyl acetate may be used alone or in combination of two or more.

In the present invention, the extract extracted with hot water or chilled extract is filtered to remove floating solid particles, for example, using nylon or the like to filter the particles, or after filtration using a freeze filtration method, etc. It can be used by drying it using spray drying or the like.

In the composition of the present invention, since each of the seed, leaf, and flower extracts of the Mugunghwa extract are effective for skin moisturizing, the Mugunghwa extract obtained by extracting any one selected from seeds, leaves, and flowers may be used.

In the pharmaceutical composition for the treatment or prevention of liver disease of the present invention, the Mugunghwa extract is characterized in that the Mugunghwa extract reduces the production of reactive oxygen species (ROS) in liver cells, and reduces lactate dehydrogenase (LDH) secretion. In an embodiment of the present invention, it was confirmed that the treatment of the Mugunghwa extract effectively inhibits the generation of active oxygen (ROS) in HepG2 cells, thereby suppressing oxidative stress and protecting the hepatocytes (see FIG. 4 ). In addition, when the liver cell membrane is damaged, lactate dehydrogenase (LDH) inside the liver cells is discharged to the outside. In particular, in the experimental group treated with Mugunghwa extract at a concentration of 100ug/ml, LDH secretion at a level similar to that of the untreated negative control group was measured. was possible (see FIG. 5).

In the pharmaceutical composition for the treatment or prevention of liver disease of the present invention, the Mugunghwa extract is characterized in that it inhibits the accumulation of fat inside the liver cells and has antioxidant activity. In the example of the present invention, it was confirmed that the Mugunghwa extract had antioxidant activity by significantly increasing the intracellular GSH content (see FIG. 8), and it was confirmed that the fat absorption in the hepatocytes was significantly reduced, improvement of non-alcoholic liver disease, It was confirmed that it can be usefully used for prevention or treatment (see FIG. 12 ).

In the pharmaceutical composition for the treatment or prevention of liver disease of the present invention, the Mugunghwa extract is a biomarker (biomarker) of liver function abnormality GOT (Glutamate Oxaloacetate Transaminase), GPT (Glutamate Pyruvate Transaminase), γ-GTP (γ-glutamyl transpeptidase) And it is characterized in that the blood content of LDH (lactate dehydrogenase) is reduced. In an embodiment of the present invention, it was confirmed that the Mugunghwa extract significantly reduced the blood content of the liver function abnormal biomarkers in the experimental animal model of alcoholic liver disease and the experimental animal model of acute liver failure (Table 1 and Table 2) Reference).

In the pharmaceutical composition for the treatment or prevention of liver disease of the present invention, the liver disease is impaired liver function, liver disorder, hepatitis, hepatotoxicity, cholestasis, fatty liver, cirrhosis, liver ischemia, alcoholic liver disease, liver abscess, hepatic coma, hepatic atrophy And it may be any one or more selected from the group consisting of liver cancer, but is not limited thereto, and preferably may be any one or more selected from the group consisting of impaired liver function, hepatotoxicity, fatty liver, liver cirrhosis, and alcoholic liver disease.

In addition, the term "improving liver function" used in the present invention means preventing liver function deterioration through inhibition of liver cell death or enhancement of activity. Specifically, it helps to improve hangover, fatigue, loss of appetite, abdominal distension, and headache, and reduces the risk of diseases such as jaundice, hepatitis, hepatic coma, ascites, platelet reduction, alcoholic fatty liver, non-alcoholic fatty liver, and liver fibrosis include functions.

In the present invention, "treatment" refers to any action in which the symptoms of liver disease are improved or beneficial by administration of the composition, and "prevention" refers to any action that inhibits or delays the onset of liver disease by administration of the composition. .

The pharmaceutical composition of the present invention comprises a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. it's not going to be The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like, in addition to the above components. Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, or the like.

A suitable dosage of the pharmaceutical composition of the present invention is variously prescribed according to factors such as formulation method, administration method, age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and reaction sensitivity of the patient. can be The daily dose of the pharmaceutical composition of the present invention is, for example, 0.001-100 mg/kg.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person of ordinary skill in the art to which the present invention pertains. or may be prepared by incorporation into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension, syrup, or emulsion in oil or an aqueous medium, or may be in the form of an extract, powder, powder, granule, tablet or capsule, and may additionally include a dispersant or stabilizer.

According to another aspect of the present invention, the present invention provides a method for preventing or treating liver disease, comprising administering a pharmaceutical composition for the treatment and prevention of liver disease to individuals other than humans. The liver disease and administration method of the present invention are as described above. As used herein, the term “administration” means introducing a given substance into a subject by an appropriate method. As used herein, the term "individual" refers to all animals, such as rats, mice, and livestock, except for humans, which have or may develop liver disease.

In another aspect, the present invention can inhibit or control liver disease by administering a pharmaceutical composition for the treatment and prevention of liver disease comprising the Mugunghwa extract as an active ingredient according to the present invention. It provides a method of inhibiting liver damage in a subject, comprising administering to the subject a pharmaceutical composition for the treatment and prevention of liver disease, including In the present invention, the term "subject" refers to a mammal including, but not limited to, cattle, dogs, pigs, chickens, sheep, horses, and humans. In addition, preferably, the administration of the composition containing the Mugunghwa extract as an active ingredient may be intraperitoneal or intravascular administration, direct administration to a lesion, or administration in a synovial cavity of a joint.

According to another aspect of the present invention, the present invention may be provided in the form of a food composition for improving and protecting liver health comprising an extract of Mugunghwa as an active ingredient. The improvement of liver function with the Mugunghwa extract is as described above, and the term 'liver protection' is used in the present specification with the same meaning as improvement of liver function, and preventing liver function deterioration through inhibition of liver cell death or enhancement of activity. it means. The food composition may be used in the form of a health functional food, but is not limited thereto.

The food composition of the present invention may be included in the form of an extract of Mugunghwa or a processed product thereof. In addition, the composition may include a food pharmaceutically acceptable food supplement additive in addition to the active ingredient.

In the present invention, "food supplement additive" means a component that can be added auxiliary to food, and is added to the manufacture of health functional food of each formulation, and those skilled in the art can appropriately select and use it. Examples of food supplement additives include various nutrients, vitamins, minerals (electrolytes), synthetic flavoring agents and flavoring agents such as natural flavoring agents, coloring agents and fillers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners , pH adjuster, stabilizer, preservative, glycerin, alcohol, carbonation agent used in carbonated beverages, etc., but the above examples are not limited to the type of food supplement additive of the present invention.

The food composition of the present invention may include a health functional food. In the present invention, "health functional food" refers to food manufactured and processed in the form of tablets, capsules, powders, granules, liquids and pills using raw materials or ingredients useful for the human body. As used herein, the term “functionality” refers to obtaining useful effects for health purposes such as regulating nutrients or physiological effects on the structure and function of the human body, and specifically refers to liver function improvement or liver protection function. The health functional food of the present invention can be prepared by a method commonly used in the art, and at the time of manufacture, it can be prepared by adding raw materials and components commonly added in the art.

In addition, if the formulation of the health functional food is also recognized as a health functional food, it can be prepared without limitation. The food composition of the present invention can be prepared in various forms, and unlike general drugs, it has the advantage that there are no side effects that may occur when taking the drug for a long time using food as a raw material, and has excellent portability, so it protects the liver Alternatively, it can be taken as an adjuvant to enhance the effect of improving liver function.

There is no limitation on the form that the health functional food of the present invention can take, and it may include any food in a conventional sense, and may be used interchangeably with terms known in the art, such as functional food. In addition, the health functional food of the present invention can be prepared by mixing known additives with other suitable auxiliary ingredients that may be included in the food according to the selection of those skilled in the art. Examples of foods that can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages and There are vitamin complexes and the like, and it can be prepared by adding the extract of Mugunghwa according to the present invention as a main component to juice, tea, jelly, juice, and the like. Also included are foods used as feed for animals.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a composition for improving and protecting liver health comprising an extract of Mugunghwa as an active ingredient.

(b) Mugunghwa extract of the present invention is not toxic to the human body, relieves oxidative stress, has an effect of directly protecting hepatocytes, directly relieves cytotoxicity of acetaldehyde, and free radicals (ROS) It effectively inhibits production, inhibits liver cell membrane damage, has antioxidant activity, and at the same time significantly reduces fat absorption in hepatocytes, thereby having an excellent effect in improving liver function and protecting the liver.

(c) In addition, the composition of the present invention using a natural plant extract is not only very safe for the human body, but also has excellent stability, so that it can be usefully used for the purpose of improving liver function or protecting the liver in the fields of food and pharmaceuticals.

1 is a graph showing the treatment of Mugunghwa extract by concentration, and measuring the cell viability.
FIG. 2 is a graph showing a HepG2 cell line induced by oxidative stress by treatment with ethanol, treated with Mugunghwa extract by concentration, and cell viability was measured.
3 is a graph showing the acetaldehyde-treated HepG2 cell line treated with Mugunghwa extract by concentration, and the cell viability was measured.
4 is a graph showing the amount of free radicals (ROS) produced by treating the Mugunghwa extract by concentration in the HepG2 cell line treated with 3% ethanol.
5 is a graph showing the treatment of Mugunghwa extract by concentration in the HepG2 cell line treated with 3% ethanol, and measuring the lactate dehydrogenase (LDH) concentration.
6 is a graph showing the H ₂ O ₂ treated HepG2 cell line treated with Mugunghwa extract by concentration, and the cell viability was measured.
7 is a graph showing the H ₂ O ₂ treated HepG2 cell line by treating the Mugunghwa extract by concentration, and measuring the MDA content, which is an indicator of lipid peroxidation.
8 is a graph showing the H ₂ O ₂ treated HepG2 cell line treated with Mugunghwa extract by concentration, and the GSH content was measured.
9 is a graph showing the D-GalN (D-galactosamine)-treated HepG2 cell line treated with Mugunghwa extract by concentration, and cell viability was measured.
10 is a graph showing the D-GalN (D-galactosamine)-treated HepG2 cell line treated with Mugunghwa extract by concentration, and the LDH (lactate dehydrogenase) concentration was measured.
11 is a graph showing the D-GalN (D-galactosamine)-treated HepG2 cell line treated with Mugunghwa extract by concentration, and the amount of active oxygen (ROS) produced was measured.
12 is a graph showing a free fatty acid (FFA)-treated HepG2 cell line treated with Mugunghwa extract and measuring the intracellular fat content.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention by these examples.

Example 1. Preparation of Mugunghwa extract

0.5 kg of Mugunghwa outpost was washed with purified water and dried, then pulverized and finely divided to obtain a powder. After adding 10 times the weight of ethanol to the powdered Mugunghwa, the mixture was extracted with an ethanol solvent while stirring overnight, filtered, and the supernatant was freeze-dried to obtain an extract.

Example 2. Verification of the effect of Mugunghwa extract on cell growth

The effect of Mugunghwa extract on the growth of human immortalized keratinocytes (HaCaT) was confirmed by the following method.

First, human keratinocytes were inoculated into a 24-well cell culture dish in DMEM (Dulbeccos modified Eagles medium) medium containing 10% FBS (fetal bovine serum, Cambrex) at a concentration of 1 × 10 ⁵ and then for 24 hours. , 37° C., 5% CO ₂ Incubated under humid conditions. Thereafter, the medium was removed and the Mugunghwa extract of Example 1 diluted with serum-free DMEM medium was treated at different concentrations (10 and 50 μg/mL) and cultured for 24 hours. 24 hours later, 1 mg/mL of MTT was treated, and 2 hours later, formazan generated by MTT treatment in cells was dissolved in DMSO and absorbance was measured at 570 nm.

As a result of the experiment, when human keratinocytes were treated with Mugunghwa extract, it was confirmed that the Mugunghwa extract did not significantly affect the cell viability of human keratinocytes. (See Fig. 1).

Example 3. Hepatoprotective activity against alcoholic oxidative stress

After ethanol treatment on hepatocytes, hepatotoxicity was induced, and the activity of Mugunghwa extract was verified to protect hepatocytes.

HepG2 cells, a human liver cancer cell line, were seeded to a concentration of 5×10 ⁴ cells/well in a 24-well plate and cultured for 24 hours. After diluting 900 μl of medium and Mugunghwa extract, 100 μl of each concentration (10, 50, and 100 μg/ml, respectively) was added, incubated for 2 hours, then treated with 300 mM ethanol and cultured for 24 hours. After repeating the above operation once every day for 3 days, the medium was removed, washed twice with PBS, replaced with a fresh medium, and 50 uL of MTT solution was added, followed by incubation for 2 hours. After removing the medium and dissolving the formazan crystals formed in the cells by adding 500ul of DMSO, the absorbance was measured at 540 nm using a microplate reader (BIORAD 680, BIORAD, USA).

As a result of the experiment, the ethanol treatment reduced the survival rate of HepG2 cells to half, but the treatment of the Mugunghwa extract increased the HepG2 cell viability in a concentration-dependent manner. In particular, in the experimental group treated with Mugunghwa extract at a concentration of 100ug/ml, cell viability equal to or greater than that of the ethanol-free group was observed.

Through the above results, it was confirmed that the Mugunghwa extract of the present invention has an effect of relieving alcoholic oxidative stress and directly protecting hepatocytes (see FIG. 2 ).

Example 4. Hepatoprotective activity against acetaldehyde

After acetaldehyde treatment on hepatocytes, hepatotoxicity was induced, and the activity of Mugunghwa extract was verified to protect hepatocytes.

HepG2 cells, a human liver cancer cell line, were seeded to a concentration of 5×10 ⁴ cells/well in a 24-well plate and cultured for 24 hours. Dilute the Mugunghwa extract and dilute it by concentration (10, 50, 100ug/ml, respectively), process 100 μl, incubate for 24 hours, change the medium, and then treat with 4 mM acetaldehyde and incubate for 24 hours did. After repeating the above operation once every day for 3 days, the medium was removed, washed twice with PBS, replaced with a fresh medium, and 50 μl of MTT solution was added, followed by incubation for 2 hours. After removing the medium and dissolving the formazan crystals formed in the cells by adding 500ul of DMSO, the absorbance was measured at 540 nm using a microplate reader (BIORAD 680, BIORAD, USA).

As a result of the experiment, acetaldehyde treatment reduced the survival rate of HepG2 cells to a level of 60%, but a concentration-dependent increase in HepG2 cell viability was observed with the treatment of Mugunghwa extract. In particular, in the experimental group treated with Mugunghwa extract at a concentration of 100ug/ml, cell viability equal to or higher than that of the acetaldehyde untreated group was observed.

Through the above results, it was confirmed that the Mugunghwa extract of the present invention has the effect of directly resolving the cytotoxicity of acetaldehyde and protecting the hepatocytes (see FIG. 3 ).

Example 5. Active oxygen (ROS) scavenging ability

HepG2 cells, a human liver cancer cell line, were treated with 3% ethanol for 30 minutes to induce an increase in intracellular reactive oxygen species (ROS) concentration. Thereafter, the Mugunghwa extract prepared in Example 1 was diluted to different concentrations (10, 50, and 100 ug/ml, respectively), treated with 100 μl, and the active oxygen (ROS) scavenging activity in HepG2 cells was measured.

After dispensing 100 μl of 30 μM DCFH-DA (Dichlorofluorescein diacete) per well of the experimental plate, it was treated at 37° C. for 30 minutes, and fluorescence reader (SpectraMax M5) was used at 485/538 nm (excitation/emission). The fluorescence value was measured and compared.

As a result of the experiment, the production of active oxygen (ROS) in HepG2 cells increased by about 65% by treatment with 3% ethanol, but a concentration-dependent decrease in the production of active oxygen (ROS) in HepG2 cells was observed with the treatment of Mugunghwa extract. became In particular, in the experimental group treated with the Mugunghwa extract at a concentration of 100ug/ml, the amount of active oxygen (ROS) produced was measured, which was reduced by 35% compared to the ethanol-treated control group.

Through the above results, it was confirmed that the Mugunghwa extract of the present invention effectively inhibits the production of free radicals (ROS) in liver cells, thereby suppressing oxidative stress and protecting the liver cells (see FIG. 4 ).

Example 6. LDH concentration increase inhibitory activity in hepatocyte model

When toxicity was caused to hepatocytes, the content of lactate dehydrogenase (LDH) secreted into the medium was measured to measure the protective effect of the Mugunghwa extract of the present invention against alcohol-induced liver cell membrane damage.

Mugunghwa extract was diluted by concentration (10, 50, 100ug/ml, respectively) and treated with 3% ethanol in a culture medium of HepG2 cells, a human hepatoma cell line, and cultured for 24 hours. The concentration of lactate dehydrogenase) was measured.

As a result of the experiment, the control group treated with only 3% ethanol increased LDH secretion by about 30% compared to the untreated negative group, but a concentration-dependent decrease in LDH secretion was observed with the treatment of Mugunghwa extract. In particular, in the experimental group treated with Mugunghwa extract at a concentration of 100ug/ml, LDH secretion at a level similar to that of the untreated negative control group was measured. It was confirmed that it can be usefully used for the improvement, prevention or treatment of liver disease (see FIG. 5 ).

Example 7. Oxidative stress inhibitory activity of Mugunghwa extract

The effect of inhibiting oxidative stress of HepG2 cells induced by H ₂ O ₂ was measured through the MTT experiment.

Mugunghwa extract was diluted by concentration (10, 50, 100ug/ml, respectively), treated with a culture medium of HepG2 cells, a human hepatoma cell line, and cultured for 24 hours, and then treated with H ₂ O ₂ at a concentration of 200 μmol/L to apoptosis. was induced.

HepG2 cells in this state were treated with MTT stock solution and left at 37° C. for 4 hours, then purple-stained formazan was dissolved in DMSO and absorbance was measured at 570 nm. Cell viability was expressed as a % value relative to the untreated control.

6, in the group treated with H ₂ O ₂ alone, the cell viability was reduced to 50% or less, but in the group treated with H ₂ O ₂ and the Mugunghwa extract of the present invention at the same time, the cell viability of HepG2 cells increased in a concentration-dependent manner. phenomenon could be observed.

In the experimental group treated with the Mugunghwa extract of the present invention, it was confirmed that the cell viability increased to 180% at a concentration of 50ug/ml and 213% at a concentration of 100ug/ml, compared to the group treated with H ₂ O ₂ alone.

Through the above results, it was confirmed that the Mugunghwa extract of the present invention exhibited excellent hepatocellular protective activity against oxidative stress induced by H ₂ O ₂ treatment, and it was confirmed that it could be usefully used for improvement, prevention or treatment of liver disease. did.

Example 8. Confirmation of oxidative damage protection effect through measurement of MDA content

ROS (reactive oxygen species) generated by oxidative stress is mostly produced by antioxidant enzymes such as superoxide dismutase (SOD), catalase, and glutathione peroxidase (GSH-Px) in vivo. However, if the antioxidant defense system is imbalanced due to excessively generated ROS, various diseases occur and aging is accelerated. Lipid peroxidation is a lipid peroxidized by the addition of oxygen to unsaturated fatty acids, which are lipid components. This means that cells or tissue membranes are damaged from free radicals. Lipid peroxide chain reacts with free radicals in polyunsaturated fatty acids and lipoproteins of cell membranes to produce malondialdehyde (MDA) as a by-product, and MDA is used as an indicator of lipid peroxidation.

By measuring the MDA content according to the treatment of Mugunghwa extract, the protective effect of oxidative liver damage was confirmed.

Hydrogen peroxide (H ₂ O ₂ ) at a concentration of 200 μmol/L was treated as a liver damage inducer, and the extract of Mugunghwa was diluted by concentration (10, 50, 100 ug/ml, respectively) and treated with a culture medium of HepG2 cells, a human liver cancer cell line. After culturing for 24 hours, the harvested hepatocytes were put in Tris-HCl buffer (pH 7.4) and homogenized, and then MDA (malondialdehyde) and 2-Thiobarbituric acid (TBA), which are decomposition products of lipid peroxide, were heated at 100°C for 20 minutes. Absorbance was measured at 532 nm of the TBA pigment produced by the process.

Referring to FIG. 7 , it was confirmed that the lipid peroxidation in hepatocytes increased by the treatment of hydrogen peroxide (H ₂ O ₂ ) was decreased in a concentration-dependent manner by the treatment of the Mugunghwa extract.

Through the above results, it was confirmed that the Mugunghwa extract of the present invention exhibits excellent hepatocellular protection activity by preventing lipid peroxidation in hepatocytes induced by H ₂ O ₂ treatment, and can be usefully used for improvement, prevention or treatment of liver disease was confirmed.

Example 9. Confirmation of oxidative damage protection effect through measurement of GSH content

The protective effect of oxidative liver damage was confirmed by measuring the GSH content according to the treatment of Mugunghwa extract.

In the same manner as in Example 8, HepG2 cells cultured under the administration of Mugunghwa extract were washed with PBS and centrifuged to obtain a pellet, and 5% (w/v) metaphosphoric acid corresponding to 4 times its volume. acid) solution and then homogenized. After centrifugation at 3,000 g for 10 minutes, the GSH content in the supernatant was measured. Intracellular GSH content was measured using a colorimetric assay kit (Bioxytech GSH-400, OxisResearch, Burlingame, CA) according to the manufacturer's method. Absorbance was measured at 400 nm using a microplate reader (BIORAD 680, BIORAD, USA).

As a result, the intracellular GSH content was significantly reduced to 25% level by treatment with hydrogen peroxide (H ₂ O ₂ ), but the intracellular GSH content of the cells cultured in the medium treated with the Mugunghwa extract increased in a concentration-dependent manner, increasing to 100ug/ In the test group treated with Mugunghwa extract at a concentration of ml, it was found that hydrogen peroxide (H ₂ O ₂ ) was recovered to a level of 80% or more of the intracellular GSH content ( FIG. 8 ). Through this, it was confirmed that the Mugunghwa extract significantly increased the GSH content in cells, and it was confirmed that it could be usefully used for the improvement, prevention or treatment of liver disease.

Example 10. Hepatocytoprotective activity of Mugunghwa extract against D-GalN toxicity

The cytoprotective activity against D-GalN (D-galactosamine), an acute hepatotoxic substance, was measured. Mugunghwa extract (10, 50, 100 ug/ml, respectively) was treated with 50 mM D-GalN (D-galactosamine) in a culture medium of HepG2 cells, and cell viability was measured.

As a result, the viability of HepG2 cells decreased to 50% with the addition of D-GalN, but it was confirmed that the cell viability increased in a concentration-dependent manner by the addition of the Mugunghwa extract. A similar level of viable cells was measured (see FIG. 9).

In addition, the Mugunghwa extract was treated with 50 mM D-GalN (D-galactosamine) in the culture medium of hepatocytes, and after 24 hours, the concentration of lactate dehydrogenase (LDH) in the culture was measured using the LDH assay kit.

As a result of the experiment, the concentration of LDH increased about 2.5 times compared to the normal group when D-GalN, an acute hepatotoxic substance, was treated. It was confirmed that the inhibition was dependent (see FIG. 10).

In addition, the active oxygen (ROS) scavenging activity according to the treatment of Mugunghwa extract was measured in hepatocytes treated with D-GalN (D-galactosamine), an acute hepatotoxic substance. After dispensing 100 μl of 30 μM DCFH-DA (Dichlorofluorescein diacete) per well of the experimental plate, it was treated at 37° C. for 30 minutes, and fluorescence reader (SpectraMax M5) was used at 485/538 nm (excitation/emission). The fluorescence value was measured and compared.

As a result of the experiment, when D-GalN, an acute hepatotoxic substance, was treated, the concentration of reactive oxygen species (ROS) increased about 5 times compared to the normal group. It was confirmed that the increase in (ROS) production was inhibited in a concentration-dependent manner (see FIG. 11 ).

Through the above results, it was confirmed that the Mugunghwa extract of the present invention is effective in the treatment and prevention of acute liver failure disease.

Example 11. Non-alcoholic fatty liver inhibitory activity

The non-alcoholic fatty liver inhibitory activity was confirmed by measuring the fat content of hepatocytes according to the treatment of Mugunghwa extract.

Nonalcoholic fatty liver disease (NAFLD) includes simple steatosis with only excessive accumulation of fat in hepatocytes, nonalcoholic steatohepatitis (NASH) with hepatocellular necrosis and inflammation and fibrosis, and more advanced It refers to a group of diseases including the form of liver cirrhosis.

It has been suggested that, as a cause of nonalcoholic fatty liver disease, direct liver damage and accumulation of fat in the liver due to excessive free fatty acid (FFA) in the body play an important role (Feldstein AE et al., Free fatty acids promote hepatic lipotoxicity by stimulating TNF-alpha expression via a lysosomal pathway (Hepatology 40:185-194, 2004).

HepG2 cells were seeded at a concentration of 2×10 ⁴ cells/well in a 24-well plate and cultured for 24 hours. After treatment with free fatty acid (FFA) at a concentration of 1 mM, the Mugunghwa extract was diluted by concentration (10, 50, and 100 ug/ml, respectively), treated with 100 μl, and cultured for 24 hours, and then the medium was removed.

After washing twice with PBS, 900 μl and 100 μl of 3 mM oleic acid dissolved in 1% BSA solution were added and cultured for 24 hours, followed by Oil Red O (ORO) staining. After removing the medium from each well and washing twice with ice PBS, 200 μl of 10% formalin was treated for 1 hour, washed twice with distilled water, and 200 μl of ORO working solution was treated at room temperature. The reaction was carried out for 2 hours. Remove the ORO solution, wash twice with distilled water to remove unstained ORO, dissolve the dyed ORO by adding 300 μl of isopropanol, and then transfer 100 μl to a 96 well plate in a microplate reader (microplate). The absorbance was measured at 490 nm using a reader, BIORAD 680, BIORAD, USA). Results are expressed as a percentage of the untreated negative control group. As a positive control group, oltipraz (a liver X receptor (LXR) antagonist) used as a treatment for nonalcoholic fatty liver was treated at a concentration of 5ug/mL.

As a result of the experiment, the intracellular fat content was significantly increased to more than 60% by treatment with free fatty acid (FFA), but the intracellular fat content of the cells cultured in the medium treated with the Mugunghwa extract decreased in a concentration-dependent manner, In the 50ug/ml Mugunghwa extract treatment group, it was found to be reduced to a level similar to that of the Oltipraz treatment group, which is a positive control group, and in the 100ug/ml Mugunghwa extract treatment group, a 27% decrease in fat content was measured compared to the positive control group (Fig. 12) Reference). Through these results, it was confirmed that Mugunghwa extract significantly reduced fat absorption in hepatocytes, and it was confirmed that it could be usefully used for improvement, prevention or treatment of non-alcoholic liver disease.

Example 12. Evaluation of liver function improvement activity in an experimental animal model of alcoholic liver disease

When liver function declines, GOT (Glutamate Oxaloacetate Transaminase), GPT (Glutamate Pyruvate Transaminase), and γ-GTP (γ-glutamyl transpeptidase) rise. varicose veins, thrombocytopenia), loss of appetite, abdominal bloating (back), headache, and bleeding gums.

The liver function improvement according to the treatment of Mugunghwa extract was evaluated in an alcoholic liver disease animal model. The experiment was conducted in the following way.

First, 35 6-week-old experimental animals (SD rats, males) were purchased and adapted for 1 week under conditions of 22±1° C., 55±3% humidity, and 12 hours of light/dark cycle (am/pm 8 o’clock). At 10 am every day for 10 days, sterile physiological saline (saline, Control, C) was administered to the normal group (N, normal) and alcohol-treated group, and UDCA (ursodeoxycholic acid, Positive Control, PC) 600㎍/ A kg BW (body weight) dose was orally administered to the Mugunghwa extract treated group at 50 and 100mg/kg BW doses, respectively. At 1 PM, the normal group was orally administered with physiological saline (saline) and the remaining treatment group 25% ethanol (alcohol) at a dose of 2 ml/kg BW. Body weight was measured every 2 days from the start of the experiment, and after 18 hours of fasting before the end of the experiment, the animals were sacrificed and blood was collected.

Body weight and liver weight were measured for each group, and the contents of GOT, GPT, γ-GT and LDH, which are biomarkers of liver function abnormality in blood, were measured and shown in Table 1 below.

[Table 1]

As a result of the measurement, there was no significant difference in body weight change in each treatment section during the experimental period except for the 100 mg/kg Mugunghwa extract treatment group. Meanwhile, in the liver weight measurement result, the liver weight increased by about 10% compared to the normal group in the negative control group administered with saline. , This liver weight loss was measured similarly to the positive control group administered with UDCA (ursodeoxycholic acid).

In addition, as a result of measuring GOT, GPT, γ-GT and LDH, which are biomarkers of liver function abnormality in the blood, GOT and It was found that the GPT values were significantly increased by about 1.5 to 2 times, respectively.

On the other hand, the positive control group (UDCA) and the treatment group fed with Mugunghwa extract showed significantly reduced GOT, GPT and γ-GT levels compared to the negative control group (Control), and similar values to the normal group were measured. In addition, the LDH level in the 50 mg/kg Mugunghwa extract treatment group did not differ significantly from the negative control group, but it was confirmed that it was significantly reduced in the 100 mg/kg treatment group.

Through the above results, it was confirmed that the Mugunghwa extract of the present invention protects hepatocytes and can be usefully used for improvement, prevention or treatment of alcoholic liver disease.

Example 13. Evaluation of liver function improvement activity in an experimental animal model of acute liver failure

Hepatoprotective activity of Mugunghwa extract was measured in an animal experiment in which acute liver failure disease was induced by treatment with D-GalN (D-galactosamine). The experiment was conducted in the following way.

First, 35 6-week-old experimental animals (SD rats, males) were purchased and adapted for 1 week under conditions of 22±1° C., 55±3% humidity, and 12 hours of light/dark cycle (am/pm 8 o’clock). At 10:00 am every day for 7 days, sterilized physiological saline (saline, Control, C) was administered to the normal group (N, normal) and alcohol-treated group, and UDCA (ursodeoxycholic acid, Positive Control, PC) 600㎍// to the positive control group. A kg BW (body weight) dose was orally administered to the Mugunghwa extract treated group at 50 and 100mg/kg BW doses, respectively.

After induction to increase liver homeostasis, at the end of the test, D-GalN (400 mg/kg) was administered intraperitoneally to induce acute liver disease. After anesthesia with ether for 6 hours, blood was collected from the heart and liver was removed, and body weight and liver weight were measured for each group. GOT, GPT, γ-GT and LDH was measured and shown in [Table 2] below.

[Table 2]

As a result of the measurement, there was no significant difference in the weight change of each treatment section during the experimental period. On the other hand, in the liver weight measurement results, the liver weight increased by about 30% compared to the normal group in the negative control group administered with saline. , This liver weight reduction was measured to be further reduced by 7% to 8% even compared to the positive control group administered with UDCA (ursodeoxycholic acid).

In addition, as a result of measuring the contents of GOT, GPT, γ-GT and LDH, which are biomarkers of liver function abnormality in the blood, the negative control group induced acute liver failure by D-GalN (D-galactosamine) treatment than the normal group. (Control) was measured to increase significantly, and it was confirmed that the positive control group (UDCA) and the treatment group fed with Mugunghwa extract were lowered. In particular, in the 100 mg/kg Mugunghwa extract treated group, values equal to or less than that of the positive control group (UDCA) were confirmed, and through these results, it was confirmed that the Mugunghwa extract of the present invention can be usefully used for improvement, prevention or treatment of acute liver failure disease. could

As described above in detail a specific part of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereto.

Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (7)

Contains Mugunghwa extract as an active ingredient,
Characterized in inhibiting apoptosis of hepatocytes under alcoholic oxidative stress or acetaldehyde conditions,
A pharmaceutical composition for the treatment or prevention of alcoholic fatty liver disease. delete delete The method of claim 1,
The Mugunghwa extract was found to reduce the blood content of Glutamate Oxaloacetate Transaminase (GOT), Glutamate Pyruvate Transaminase (GPT), γ-glutamyl transpeptidase (γ-GTP) and lactate dehydrogenase (LDH), which are biomarkers for liver function abnormalities. A pharmaceutical composition for the treatment or prevention of alcoholic fatty liver disease, characterized in that. delete Contains Mugunghwa extract as an active ingredient,
Characterized in inhibiting apoptosis of hepatocytes under alcoholic oxidative stress or acetaldehyde conditions,
A food composition for improving and protecting liver health against alcoholic fatty liver disease. A health functional food comprising the food composition according to claim 6 .

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