KR101187815B1 - Pharmaceutical Compositions for Preventing or Treating Liver Disorders Comprising Oenanthe javanica Extracts as an Active Ingredient - Google Patents

Abstract

Fluoride chlorogenic acid (Bluminic acid extract), Burmese extract (BM), Fluoride SOD1 (superoxide dismutase 1) Purified product (BMS), Butyl isoferulic acid (isoferulic acid) or gallic acid (gallic acid) as an active ingredient A composition for preventing or treating liver disorders. The radish extract and radish SOD1 purified product of the present invention are characterized by peroxidation of alcohol-derived liver lipids, levels of hepatic glutathione (GSH), levels of alcohol-derived liver triglycerides (TGs) or phosphorylated ACC. It reduces the levels of (acetyl-CoA carboxylase), increases the expression of CYP2E2 (cytochrome P450 2E2) and the phosphorylation of AMPK (AMP-activated protein kinase). Furthermore, the vulcanized chlorogenic acid, vulcanized SOD1 purified product (BMS), vulcanized isoferulic acid and vulgaric gallic acid (gallic acid) identified from the radish fermented extract of the present invention are alcoholic dehydrogenase (ADH). ) And increase the activity of ALDH (aldehyde dehydrogenase). Therefore, the composition of the present invention comprising the above-mentioned fermented radish fermented extract and the compound identified therefrom as an active ingredient can be usefully applied to the prevention or treatment of liver disease, and can also be used as a food composition for improving hangover.

Description

Pharmaceutical Compositions for Preventing or Treating Liver Disorders Comprising Oenanthe javanica Extracts as an Active Ingredient}

The present invention relates to a flame extract (BM) having an alcohol resolution and an active ingredient thereof.

Alcohol is one of the oldest drugs that humans have used since civilization. Large amounts of alcohol consumption lead to fatal problems in the body, including alcoholic liver diseases (ALDs). ALD is the most common liver disease in the UK, affecting more than 20,000 people each year. Many pathways are thought to be involved in ALD, including oxidative stress and mitochondrial damage. Ethanol is metabolized to acetaldehyde in the body by the catalysis of enzymes: Acetaldehyde is oxidized to acetate and then converted to carbon dioxide through the citric acid cycle. Ethanol also affects the immune system and alters cytokine production, increasing levels of hepatic triglyceride and lipid peroxidation and reducing hepatic glutathione (GSH) content. GSH not only functions as a regenerating factor of free radical scavengers and -tocopherols, but also plays an important role in maintaining protein sulfhydryl groups.

Oxidative stress is known to play an important role in the pathogenesis of ethanol-induced liver damage. Induction of CYP2E1 by ethanol is key and pathological changes in ethanol-induced liver damage are associated with CYP2E1 levels. CYP2E1 expression levels are induced by changes in alcohol-dependent metabolism in early liver damage such as hepatitis and fatty liver. In addition, CYP2E1 is well known to be involved in the activation of many chemicals related to toxic agents and carcinogens. Natural compounds that reduce the enzymes that activate the above-mentioned chemicals can be considered preferred candidates for defense against chemically induced toxicity. Recently, herbs have attracted attention as source materials for the development of healthful foods (physiologically functional foods) and drugs. The use of herbal medicines derived from plant extracts is increasing to treat a wide range of clinical diseases.

Oenanthe javanica , also called stone parsley or field parsley, is a perennial herbaceous genus, widely cultivated and used in folk medicine, and its leaves and stems are lowered in Korean herbal medicine, lowering blood pressure, fever, diuresis, blood, hemostasis, tonic, It is one of the medicinal plants used for the treatment of poison and pneumonia (Chinese medicine dictionary, Shanghai Science and Technology Press, p1332-1333, 1981, Ph.D., Ph.D., p306, 1980). In particular, the vulgaris contains high amounts of potassium, calcium, vitamin C and carotene and vegetable fiber, which are converted to vitamin A in the body, and especially SOD, a type of chlorophyll and antioxidant enzymes. In addition, essential oils that give off the unique aroma and flavor of the radish parsley not only boosts the taste, but also clears the mind and cleanses the blood. Specifically, appetite enhancement, blood pressure lowering, diuresis, detoxification is excellent and contains a lot of useful components such as the liver, small intestine, large intestines, such as the digestive heart and heart, kidneys.

However, the functions and mechanisms of protection of hepatocytes and prevention of liver damage of SOD1 (superoxide dismutase 1) of the vulgaris or the vulgaris have not been revealed, and it has not been used as a functional health-oriented food material such as beverages. It never happened.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have tried to find natural compounds and active ingredients thereof that can prevent or treat liver damage. As a result, the inventors of the present invention have been directed to the radish extract (BM), preferably the radish chlorogenic acid, the radish fermented extract, the radish SOD1 (superoxide dismutase1) purified product (BMS), the radish isoferulic acid or radish The present invention was completed by confirming that the pharmaceutical composition including gallic acid as an active ingredient can prevent or inhibit alcohol-induced liver damage.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for preventing or treating liver disease.

Another object of the present invention to provide a food composition for improving the hangover.

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 an aspect of the present invention, the present invention is a fluoride chlorogenic acid (chlorogenic acid), fern fermented extract, bulmus extract (BM), fluoride SOD1 (superoxide dismutase 1) purified product (BMS), fluoride isoperulic acid (isoferulic acid) ) Or a composition for preventing or treating liver disorders comprising gallic acid as an active ingredient.

According to another aspect of the present invention, the present invention provides a pharmaceutical composition comprising (a) a pharmaceutically effective amount of the composition described above; And (b) provides a pharmaceutical composition for preventing or treating liver disease comprising a pharmaceutically acceptable carrier.

According to another aspect of the present invention, the present invention is a radish chlorogenic acid (chlorogenic acid), radish fermented extract, radish extract (BM), radish SOD1 purified product, radish isoperulic acid (isoferulic acid) or radish gallic acid (gallic It provides a food composition for improving the hangover containing acid) as an active ingredient.

The present inventors have tried to find natural compounds and active ingredients thereof that can prevent or treat liver damage. As a result, the present inventors include as an active ingredient a radish extract (BM), preferably a radish SOD1 purified product, a radish chlorogenic acid, a radish isofulic acid or a gallic acid as an active ingredient. It has been confirmed that the pharmaceutical composition can prevent or inhibit alcohol-induced liver damage.

It has long been used as a valuable medicine because it protects the liver and has excellent detoxification and tonic effects. Traditionally, fire beaks, known as liver-improving medicinal herbs, are our traditional wild grasses that grow wild at the foot of the mountain or in the fields without water. Unlike ordinary water parsley, which grows in water, it has been used for a long time because it receives enough sunlight and grows red light on its stem, and has a strong fragrance and high sugar content. Buttercup's anti-cancer effect is associated with quercetin and kaempferol, a type of green flaming pigment flavonoid. Quercetin and camphorol inhibit the production of reactive oxygen species in the body that promote aging and mutation of cells, thereby preventing breast cancer, colon cancer, ovarian cancer, gastric cancer, bladder cancer, and prostate cancer.

The present inventors have shown a new mechanism of action on protecting hepatocytes and preventing liver damage of the buds, and isolated / identified their active ingredients. Buttercup extract of the present invention can be prepared according to a method that can be easily carried out by those skilled in the art.

According to the present invention, the radish fermented extract (BM) of the present invention was obtained primarily as a hot water extract, separated / purified using a suitable resin (eg, HP-20 resin), and stored by freeze drying. When the radish extract used in the composition of the present invention is obtained by treating the extracting solvent, various extracting solvents may be used. Preferably, a polar solvent or a nonpolar solvent can be used. Suitable polar solvents include (a) water, (b) methanol, (c) CH ₂ Cl ₂ , (d) ethanol acetate (EtOAc), (e) n- BuOH, (f) acetic acid, (g) DMFO ( dimethyl-formamide) and (h) dimethyl sulfoxide (DMSO). Suitable as nonpolar solvents are acetone, acetonitrile, ethyl acetate, methyl acetate, fluoroalkane, pentane, hexane, 2,2,4-trimethylpentane, decane, cyclohexane, cyclopentane, diisobutylene, 1- Pentene, 1-chlorobutane, 1-chloropentane, o -xylene, diisopropyl ether, 2-chloropropane, toluene, 1-chloropropane, chlorobenzene, benzene, diethyl ether, diethyl sulfide, chloroform, dichloro Methane, 1,2-dichloroethane, anneal, diethylamine, ether and THF.

More preferably, the extraction solvent used in the present invention includes (a) water, (b) methanol, (c) CH ₂ Cl ₂ , (d) ethanol acetate (EtOAc) and (e) n -BuOH.

As used herein, the term extract has a meaning commonly used as a crude extract in the art as described above, but also broadly includes a fraction additionally fractionating the extract. In other words, the extract is not only obtained by using the above-described extraction solvent (for example, water), but may also include those obtained by additionally applying a purification process. For example, a variety of chromatography (eg, silica gel, RP-C ₁₈ , Sephadex LH 20 for separation according to fraction, size, charge, hydrophobicity or affinity obtained by passing the extract through an ultrafiltration membrane having a constant molecular weight cut-off value). Fractions obtained through various purification methods additionally performed, such as separation by Licroprep RP-C ₁₈ , Toyopearl HW 40, Preparative HPLC, etc.) may also be included in the BM of the present invention.

According to a preferred embodiment of the present invention, the BM of the present invention has alcohol degradability.

According to a preferred embodiment of the present invention, the BM of the present invention inhibits collagen production and reduces the expression of matrix metalloprotease (Matirix mataloprotease (MMP)).

According to a preferred embodiment of the present invention, the BM of the present invention has a protective effect of hepatocytes (more preferably hepatic stellate cells) against oxidative stress (eg, inhibition of production of reactive oxygen species and DNA damage or hepatic lipid peroxidation). Decreases).

More specifically, the BM of the present invention inhibited the production of concentration-dependent reactive oxygen species (ROS) in chemical (eg CCl ₄ or t-BHP) -derived liver fibrosis animal models ( Note: Figure 5a).

Carbon tetrachloride (CCl ₄ ) is a sample that damages the liver, and is widely used to study liver damage mainly by direct administration to rodents such as mice. Carbon tetrachloride reacts with metabolic enzymes, such as cytochrome P450, to produce free radicals CCl ₃ that cause oxidation of hepatic triglycerides and membrane phospholipids, which combine with oxygen to form lipid peroxides. In addition, these peroxides destroy several functions of the liver and cause damage to liver tissue (Chang. JM et. Al., Drug and chemical toxicology, 6. 443-453, 1983).

t-BHP ( tert- butylhydroperoxide) is an organic peroxide that is widely used in various oxidation processes and is generally provided as an aqueous solution of 69-70%. T-BHP, an organic free radical, artificially induces lipid peroxidation, leading to oxidative stress. In other words, t-BHP induces a severe oxidative stress response by causing destabilization due to peroxidation of membrane lipids and interconnection of membrane proteins and increasing the action of phospholipid analogs, thereby degrading the normal function of cell membranes.

As used herein, the term reactive oxygen species (ROS) means that oxygen, which is essential for organic respiration organisms, is incompletely reduced or grown in peptides during enzymatic and most electron transport or energy metabolism processes in cells. Oxygen by-products generated by stimuli of factors, cytokines and various actions. In other words, reactive oxygen species have a free radical (a form in which an atom or molecule has electrons unpaired in its outer orbit), which means that it is unstable, and thus has strong activity.

Reactive oxygen species are sometimes referred to as harmful oxygen because excessive production leads to toxicity to the living organism, that is, oxidative stress. By oxidizing huge molecules (proteins, lipids, etc.) in the cell, it destroys the homeostasis of cells and kills the cells, causing fatal damage in cell tissues, cancer, muscular dysplasia, Alzheimer's disease, Parkinson's disease, ischemic diseases This is because it is related to the causes of various degenerative diseases such as atherosclerosis and general aging.

For example, reactive oxygen species include lipid peroxide (lipid) in addition to superoxide anion radical (O ^{2 −} ), hydrogen peroxide (H ₂ O ₂ ), hydroxy radical (OH), and the like. peroxide), Li trick oxide (Nitric oxide: NO), peroxy nitrite _{^{(peroxinitrite: NO 3 2 -)}} , thiols peroxy radical (thiol peroxy radical: R-SO 2-) and the like, of H ₂ O _The main species produced by stimulation such as bivalent TGF, PDGF, and EGF, are attracting the most attention as secondary signaling substances. The reason is that it is relatively stable, less toxic, and easily diffuses and can pass through cell membranes unless it reacts with transition metals. Because.

As described above, the inventors further separated / purified BM to further separate BM fractions (eg, BMS, SOD1 (superoxide dismutase 1; SEQ ID NO: 1) -containing fractions of the radish extract; BMS-1, SOD1 monocomponent of the vulgaris extract; BMS-2, SOD2 monocomponent of the vulgaris extract; Inulin single component of BMI, Burberry extract; Oligo single component of BMO, radish extract; Fluoride shimissipuccinic acid; Chlorogenic acid; Fluoride ferulic acid; Fluoride isoferulic acid; Flaky gallic acid; And flameless scopoletin] were used to identify single active ingredients. According to a preferred embodiment of the present invention, the single component of the present invention comprises an amino acid sequence consisting of the nucleotide sequence encoding the amino acid sequence of the SOD1 or its complementary nucleotide sequence, more preferably consisting of the first sequence of the sequence listing It is apparent in the art that amino acid sequences are included and variants thereof may also be used.

According to a preferred embodiment of the present invention, the BMS (more preferably BMS-1) of the present invention is alcohol-derived hepatic lipid peroxidation, hepatic glutathione (GSH) and hepatic triglycerides (TGs) Decreases the level.

According to a preferred embodiment of the present invention, the BMS (more preferably, BMS-1) of the present invention increases the expression of CYP2E2 (cytochrome P450 2E2), increases the phosphorylation of AMPK (AMP-activated protein kinase), Decreases the level of phosphorylated ACC (acetyl-CoA carboxylase).

According to a preferred embodiment of the present invention, active ingredients (ie, active ingredients) that increase the activity of ADH (alcohol dehydrogenase) and ALDH (aldehyde dehydrogenase) are fluoride chlorogenic acid, fluoride SOD1 (superoxide) identified from BMS of the present invention. dismutase (BMS) fractions, flameless shimissipuccinic acid, flameless ferulic acid, flameless isoferulic acid and flameless gallic acid, more preferably flameless chlorogenic acid, flameless SOD1 fraction, flameless ferulic acid, flameless isoferulic acid and flameless Gallic acid, most preferably fluoride chlorogenic acid, fluoride SOD1 fraction, fluoride isoperulic acid and fluoride gallic acid.

Meanwhile, according to the present invention, the fermentation of the present invention is carried out as follows:

a) washing and pulverizing the buttercups;

b) mixing the ground fire with sugar; And

c) naturally fermenting the mixture.

In the production method of the present invention, the ratio of the pulverized and sugar pulverized in step (b) is mixed in a ratio of 1: 1-1: 2, preferably in a ratio of 1: 1, followed by fermentation for 4 to 1-2 years. It is characterized by.

In the present invention, flaming is used in the sense of including all flammables processed in natural and various forms, and the method of the present invention can be applied to these various forms of flaming. These various types of flares may differ in component content but it is apparent to those skilled in the art that the effects are the same by going through the manufacturing steps of the present invention.

The origin of the fire radish used in the present invention is not particularly limited, and for example, all domestic, US, Japanese, Himalayan, Vietnamese and Chinese products can be used.

The fire radish fermentation extract used in the present invention can be obtained through natural fermentation, it can be easily prepared by using the fire radish extract. For example, methods such as hot water extraction, organic solvent extraction, and the like may be used, and specifically, the radish fermented extract may be extracted by heating at 100 or more for 4-20 hours while stirring as necessary. In addition, the radish extract used in the present invention may further be a radish extract after the conventional cooling, filtration, concentration, and sterilization process.

Not the microorganism which can be used in the manufacture of bulminari fermentation extract of the present invention is particularly limited to known microorganisms in the art, e.g., Aspergillus (Aspergillus), and in micro-organisms, in particular Aspergillus awamori than (Aspergillus awamori ), Aspergillus ficuum ), Aspergillus niger), Aspergillus come dissimilar (Aspergillus oryzae ) and Aspergillus sojae .

The liver is one of the largest and most complex organs in the human body. The liver has a variety of functions, including: (a) production of proteins and enzymes; (b) detoxification; (c) metabolic function; And (d) control of cholesterol and blood coagulation. Since the liver is primarily responsible for alcohol metabolism, it is very sensitive to alcohol-associated damage.

The term liver function as used herein refers to normal function of the liver, for example serum proteins (eg albumin, coagulation factor, alkaline phosphatase), aminotransferases (eg alanine transaminase, aspartate trans) Aminase), 5-nucleosidase, -glutaminyltranspeptidase-like proteins, bilirubin and cholesterol; Liver metabolic functions including carbohydrate, amino acid and ammonia metabolism, hormone metabolism and lipid metabolism; Detoxification function for exogenous drugs; And hemodynamic functions including but not limited to visceral and portal hemodynamics.

According to a preferred embodiment of the present invention, the liver disease of the present invention includes acute or chronic alcoholic liver disease (ALD), more preferably hepatic steatosis, alcoholic hepatitis, liver fibrosis and cirrhosis Including, but not limited to.

The term alcoholic liver disease (ALD) herein is a serious and fatal disease resulting from alcohol abuse. ALD includes three conditions: (a) fatty liver; (b) alcoholic hepatitis; And (c) cirrhosis. Fatty liver (eg, steatosis), the most common alcohol-induced liver disease, is characterized by excessive accumulation of fat inside hepatocytes. Alcoholic hepatitis is inflammation and more serious damage to the liver, which causes the body's immune system to respond to and cause liver damage. Liver cirrhosis is when normal hepatocytes are replaced by wound tissue (ie fibrosis), and as a result the liver cannot function much of its normal function.

ALD is a major cause of liver disease in the West, while viral hepatitis is a major cause in Asia. ALD results from excessive alcohol intake. Although millions of people drink alcohol regularly, only a few excessive alcohol users have liver damage. How alcohol damages is not yet fully understood. It is known that alcohol produces toxic chemicals such as acetaldehyde, which can damage liver cells, but it is still a controversial issue why it occurs only in some excessive alcohol users. If alcohol damages the liver, its remarkable regenerative capacity does not immediately impair its function, even if about 75% of the liver is damaged. When alcohol is consumed for a long time, liver scarring known as cirrhosis or terminal alcoholic liver disease eventually develops.

The term treatment or treating herein means to obtain the desired pharmacological and / or physiological effect. The effect is to completely or partially prevent a disease or symptom and / or to completely or partially treat a disease and / or side effects that contribute to that disease. In other words, the term treatment as used herein refers to all treatments of a disease in a mammal, preferably a human, which includes: (a) Diseases that occur in subjects susceptible to disease but not yet diagnosed; To prevent; (b) inhibiting the disease, ie, arresting the development of the disease; And (c) recovering from the disease.

The term alcoholic hepatitis herein refers to acute or chronic inflammatory lesions of the liver resulting from chronic alcohol abuse. The term alcoholic hepatic (liver) fibrosis as used herein refers to the growth of liver scar tissue in the liver resulting from chronic alcohol abuse.

The term liver cirrhosis as used herein refers to all stages of development of pathological conditions, including the very early development of fibrous scar tissue to full-blown cirrhosis. Examples of diseases or conditions known to lead to cirrhosis include alcoholic liver disease, chronic viral hepatitis (B and C), chronic bile duct blockage and abnormal storage of copper (Wilson's disease) or iron (hemochromatosis). Metabolic diseases, including but not limited to. In addition, cirrhosis is caused by exposure to drugs and toxins, autoimmune processes such as autoimmune hepatitis, genetic diseases such as cystic fibrosis and alpha anti-trypsin deficiency, and obesity (eg, fatty liver and non-alcoholic steatohepatitis). May be caused. Furthermore, harmful reactions of prescription drugs, prolonged exposure to environmental toxins such as arsenic, schistosomiasis following parasitic infections, and repeated bouts of heart failure following liver congestion can all lead to cirrhosis.

The term prevention of liver cirrhosis as used herein means preventing or attenuating the damage of liver tissue, such as the accumulation of fibrous scar tissue, caused by factors known to cause cirrhosis. Factors include alcoholic liver disease, chronic viral hepatitis (types B and C), chronic bile duct blockage, Wilson's disease, hemochromatosis, exposure to drugs and toxins, autoimmune hepatitis, cystic fibrosis, alpha anti-trypsin deficiency, obesity and Schistosomiasis, including but not limited to.

Meanwhile, the composition of the present invention may be prepared as a pharmaceutical composition. Preferably, the composition of the present invention comprises (a) a pharmaceutically effective amount of the above-mentioned flaming extract of the present invention; And (b) a pharmaceutically acceptable carrier. As used herein, the term pharmaceutically effective amount means an amount sufficient to achieve the efficacy or activity of the above-mentioned flaky extract.

When the composition of the present invention is made into a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, 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 doesn't happen. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and agents are Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered orally or parenterally, and preferably applied by oral administration.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . On the other hand, the oral dosage of the pharmaceutical composition of the present invention is preferably 0.5-200 mg / kg per day on an adult basis.

The pharmaceutical compositions of the present invention may be prepared in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container. The formulation may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of extracts, powders, powders, granules, tablets or capsules, and may further comprise dispersants or stabilizers.

In addition, the composition of the present invention may be provided as a food composition. When the composition of the present invention is prepared as a food composition, as an active ingredient, as well as the extract of the parsley, it contains components that are commonly added during food production, for example, proteins, carbohydrates, fats, nutrients, seasonings and flavoring agents. It includes. Examples of the above carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And sugars such as conventional sugars such as polysaccharides such as dextrin, cyclodextrin and the like and xylitol, sorbitol, erythritol. As flavoring agents, natural flavoring agents (tauumatin, stevia extract (for example rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is prepared with a drink, in addition to the octagonal extract of the present invention, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, tofu extract, jujube extract, licorice extract, etc. may be further included. Can be.

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

(a) The present invention relates to a flameless fermented extract (BM) having an alcohol resolution.

(b) The radish fermented extract and the radish SOD1 purified product of the present invention are alcohol-induced hepatic lipid peroxidation, hepatic glutathione (GSH) levels, alcohol-induced hepatic triglycerides (TGs) Or reduces the level of phosphorylated ACC (acetyl-CoA carboxylase).

(c) In addition, the radish extract and radish SOD1 purified product of the present invention increase the expression of CYP2E2 (cytochrome P450 2E2) and phosphorylation of AMPK (AMP-activated protein kinase).

(d) Furthermore, fluoride chlorogenic acid, fluoride SOD1 purified product (BMS), vulgarisol isoferulic acid and fluoride gallic acid identified from the fluoride fermentation extract of the present invention. Increases the activity of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH).

(d) Therefore, the composition of the present invention comprising the above-mentioned fermented radish fermented extract and the compound identified therefrom as an active ingredient can be usefully applied to the prevention or treatment of liver disease, and can also be used as a food composition for improving hangovers. have.

Figure 1 shows the results of examining the cytotoxicity of the bulrush extract (BM). Treatment of several fractions of Buttercup extract showed no cytotoxicity at concentrations below 200 μg / ml. Abbreviations: BM, Burberry Extract; Inulin single component of BMI, Burberry extract; Oligo single component of BMO, radish extract; BMS, superoxide dismutase (SOD) -containing fraction of bulrush extract; BMS-1, SOD1 monocomponent of the vulgaris extract; And BMS-2, SOD2 monocomponent of the bulrush extract.
Figure 2 is the result of measuring the change in collagen synthesis in hepatic stellate cells by the BB extract. Figure 2a is the result of measuring the amount of radioisotope-labeled proline ( ³ H-proline) after BM treatment in hepatic stellate cells derived from primary cultured experimental animals. After the cells were treated with salt or treated with BM (10, 50, 100 or 200 μg / ml, oral administration), the amount of ³ H-proline was measured. The amount of ³ H-proline in the BM-treated group was significantly reduced. Values are mean ± standard deviation for the amount of ³ H-proline measured in hepatic stellate cells. * P <0.05, statistically significantly different from the control group. Figure 2b is the result of measuring the amount of hydroxyproline, a collagen precursor after BM treatment to hepatic stellate cells derived from primary cultured experimental animals. After the cells were treated with salt or treated with BM (10, 50, 100 or 200 μg / ml, oral administration), the amount of hydroxyproline was measured. The amount of hydroxyproline in the BM-treated group was significantly reduced. Values are mean standard deviation of the amount of hydroxyproline measured in hepatic stellate cells (1 × 10 ⁶ cells). * P <0.05, statistically significantly different from the control group. Figure 2c is the result of measuring the amount of hydroxyproline, a collagen precursor after treatment of BM fractions in hepatic stellate cells derived from primary cultured experimental animals. After the cells were treated with salt or with the indicated amounts of BM or BM fractions, the amount of hydroxyproline was measured. The amount of hydroxyproline was significantly reduced in the groups treated with BM (200 μg / ml, oral administration) or BM fractions (BMS4, BMS-1 or BMS-2; μg / ml, oral administration). Values are mean ± standard deviation of the amount of hydroxyproline measured in hepatic stellate cells. * P <0.05, statistically significantly different from the control group. Abbreviations: BM, Burberry Extract; BMI, Inulin Single Component of Burberry Extract; Oligo single component of BMO, radish extract; BMS 4, SOD-comprising fraction of bulrush extract; BMS-1, SOD1 monocomponent of the vulgaris extract; And BMS-2, SOD2 monocomponent of the bulrush extract. 2D and 2E show the results of staining the amount of collagen in hepatic stellate cells using Marson-Trichrome staining and immunostaining, respectively. The amount of collagen in hepatic stellate cells was significantly reduced in the group treated with fire parsley.
3A is an RT-PCR result investigating the effect of BM on the expression of matrix metalloprotease-2 (MMP-2) involved in collagen production in a chemical-induced liver fibrosis animal model. The amount of matrix metalloprotease-2 (MMP-2) was decreased in a concentration-dependent manner compared to the control group. 3B and 3C show the effects of BM in carbon tetrachloride (CCl ₄ ) -induced liver fibrosis animal model using Marson-Trichrome staining and immunostaining, respectively. Carbon tetrachloride (CCl ₄ ) -induced hepatic fibrosis was increased, and in the acupuncture treatment group, carbon tetrachloride-induced hepatic fibrosis was decreased depending on treatment concentration.
Figure 4 is a result showing the inhibitory effect of BM on oxidative stress in t-BHP ( tert -butylhydroperoxide) -treated liver fibrosis animal model. Primary cultured hepatic stellate cells induced by hepatotoxicity by t-BHP (250 M) were treated with BM of the present invention to investigate the inhibitory ability against oxidative stress by measuring hepatocyte recovery through an MTT assay. Cell survival was significantly increased in the groups treated with BM fractions (BMS 100 μg / ml; BMO 60 μg / ml; BMI 60 μg / ml; BMS 2, oral administration). Values are mean standard deviation of hepatic stellate cell viability. * P <0.05, statistically significantly different from the control group. Abbreviation: BMS, SOD-comprising fraction of radish extract; BMI, Inulin Single Component of Burberry Extract; Oligo single component of BMO, radish extract; And BMS 2, SOD2 monocomponent of the vulgaris extract.
5 is a result showing the inhibitory effect of BM on oxidative stress in hepatic stellate cells. Figure 5a is the result of measuring the fluorescence staining the amount of reactive oxygen species (ROS) DC2 (2,7-dichlorofluorescin). The amount of fluorescence was significantly reduced in the BMS-treated group at 1.0 or 5.0 μg / ml among the BM fraction-treated groups. The value is the mean value ± standard deviation for the detected fluorescence. * P <0.05, statistically significantly different from the control group. 5b shows the results of DNA damage in hepatic stellate cells. The damage of t-BHP (250 M) -induced DNA was markedly reduced by BMS treatment. The value is the mean value ± standard deviation for the detected tail movement. * P <0.05, statistically significantly different from the control group. Figure 5c is the result of histological analysis using hematoxylin-eosin (HE) staining. Liver tissues were fixed in neutral-buffered formalin for 24 hours, then processed according to the usual standard methods, embedded in paraffin and sectioned, followed by deparaffinization and rehydration. The sections were examined for morphological changes through hematoxylin and eosin staining to determine the extent of infection.
FIG. 6 shows the results of alcohol resolution of BM leaching liquor in alcohol-treated liver. As a result of quantifying the amount of alcohol present in the blood after treatment with alcohol, the amount of alcohol was less detected in the BM leaching liquor compared to the control group.
7 is a result showing the effect of BMS on liver GSH concentration. Mice were pretreated with BMS (0.5, 1.0 or 2.0 mg / kg, orally) once daily for six consecutive days. Control mice received salts. After 6 days, ethanol (50%) diluted with normal salt was administered orally to mice at a dose of 5 g / kg every 12 hours. Mice were sacrificed 18 hours after the final ethanol administration. Liver glutathione was measured as described in the experimental material. Values are mean standard deviation for four mice. # P <0.05 significantly different from control. * P <0.05 significantly different from ethanol treated group.
8 is a result showing the effect of BMS on CYP2E1 protein expression and activity. 8A is an immunoblotting result of examining CYP2E1 protein expression. Liver microsomes were obtained from mice pretreated with BMS (0.5, 1.0 or 2.0 mg / kg, oral administration) once daily for 6 consecutive days. Control mice received salts. After 6 days, ethanol diluted with normal salt was administered orally to the mice three times at a dose of 5 g / kg every 12 hours. Mice were sacrificed 18 hours after the final ethanol administration. Liver CYP2E1 protein levels were detected by immunoblotting each liver protein sample with a specific antibody. GAPDH protein was used as a loading control. 8B is a graph showing the CYP2E1 enzyme activity. CYP2E1 enzyme activity was measured as described in the experimental material. Values are mean ± standard deviation for four mice. # P <0.05 significantly different from control. * P <0.05 significantly different from ethanol treated group.
9 is a result showing the effect of BMS on liver TG content. Rats received a pair of control or alcohol-containing diets for 5 weeks. Two weeks after the start of the diet, BMS (0.5, 1.0 or 2.0 mg / kg) was treated daily for the remaining three weeks in the animals. Liver TG content was measured as described in the experimental method. Values are mean ± standard deviation for four mice. # P <0.05 significantly different from control. * P <0.05 significantly different from ethanol treated group.
10 shows the results of observing the effect of BMS on histopathological changes in liver. Rats received a pair of control or alcohol-containing diets for 5 weeks. Two weeks after the start of the diet, animals were treated with BMS (2.0 mg / kg) daily for the remaining three weeks. To assess pathological changes according to control diet (A), ethanol diet (B) or ethanol and BMS (2.0 mg / kg) diet (C), livers were obtained, fixed in formalin and stained with hematoxylin-eosin. . # P <0.05 significantly different from control. * P <0.05 significantly different from ethanol treated group.
11 is an immunoblotting result showing the effect of BMS on the phosphorylation of AMPK. Rats received a pair of control or alcohol-containing diets for 5 weeks. Two weeks after the start of the diet, animals were treated with BMS (0.5, 1 or 2.0 mg / kg) daily for the remaining three weeks. Immunoblotting against phosphorylated AMPK in liver homogenates prepared from 4 animals per treatment. Representative blots are shown in the figure: One sample per treatment was included in the blot. GAPDH protein was used as loading control. # P <0.05 significantly different from control. * P <0.05 significantly different from ethanol treated group.
12 is an immunoblotting result showing the effect of BMS on the phosphorylation of ACC. Rats received a pair of control or alcohol-containing diets for 5 weeks. Two weeks after the start of the diet, animals were treated with BMS (0.5, 1 or 2.0 mg / kg) daily for the remaining three weeks. The level of phosphorylated ACC was measured in liver homogenates prepared from 4 animals per treatment. Representative blots are shown in the figure: One sample per treatment was included in the blot. GAPDH protein was used as loading control. # P <0.05 significantly different from control. * P <0.05 significantly different from ethanol treated group.
Figure 13 is a result showing the effect on the in vitro ADH activity increase of Burberry extract (BMS).
Figure 14 is a result showing the effect on the in vitro increase in ALDH activity of Burberry extract (BMS).
FIG. 15 is a schematic diagram showing a process of separating / purifying a compound from a Burberry extract (BMS). FIG.
FIG. 16 shows the results of HPLC profiling of compounds isolated from Burberry Extract (BMS).
FIG. 17 is a result showing the effect of in vitro ADH activity increase of the compound isolated from the Burberry extract (BMS): 1, untreated group; 2, 1,000 unit / ml SOD; 3, 1 mg / ml caffeic acid; 4, 1 mg / ml Flaky Extract; 5, 1 mg / ml simicifujic acid E; 6, 1 mg / ml chlorogenic acid; 7, 1 mg / ml ferulic acid; 8, 1 mg / ml isoferulic acid; 9, 1 mg / ml gallic acid; And 10, 1 mg / ml scopoline.
FIG. 18 is a result showing the effect of the in vitro ALDH activity increase of the compound isolated from the Burberry extract (BMS): 1, no treatment; 2, 1,000 unit / ml SOD; 3, 1 mg / ml caffeic acid; 4, 1 mg / ml Flaky Extract; 5, 1 mg / ml simicifujic acid E; 6, 1 mg / ml chlorogenic acid; 7, 1 mg / ml ferulic acid; 8, 1 mg / ml isoferulic acid; 9, 1 mg / ml gallic acid; And 10, 1 mg / ml scopoline.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Materials and Experiments

chemical substance

Ethanol, thiobarbituric acid (TBA), dithionitrobenzene acid, L-ascorbic acid, phenylmethoxysulfonyl fluoride, reduced glutathione (GSH), Bradford solution and serum alanine aminotransferase (ALT) and serum aspartate amino Diagnostic kits for measuring transferase (AST) and serum albumin levels were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Lieber DeCarli liquid diet was purchased from Dyets, Inc. (Bethlehem, PA). Antibodies that specifically recognize AMPKa phospho-AMPKa, ACC and phospho-ACC were obtained from Cell Signaling (Beverly, MA), and antibodies against CYP2E1 and GAPDH were Abcam (UK) and Santa Cruz Biotechnology (Santa Cruz), respectively. , CA). All other compounds used the highest purity grade available commercially.

Flaming  extract

Thoroughly rinse and wash the buttercups with the weight of the raw buttercups washed with brown sugar (refined white sugar, CJ, Korea) and oligosaccharides (55% of fructo oligosaccharide, 100 ° Brix, Sanyang Co., Korea) as leachants. The sugar ratio was mixed 1: 1 to ferment. The fire-fermented fermented sugar was stored at room temperature for 1 year, so that useful ingredients in the raw material were eluted by osmotic pressure and spontaneous fermentation occurred. When leaching of the bulmus is completely done, the supernatant was transferred to a storage container, and the fluoride liquid extract aged for one or two years in a 4 ° C. cold storage room was used as a sample or concentrated. A methanol solution (36 L) was added thereto, followed by extraction with continuous stirring at room temperature, followed by filtration with a nonwoven fabric and reduced pressure / concentration to obtain a methanol extract (550 g). Next, the obtained methanol extract was dissolved in 80% aqueous methanol solution (4 L), followed by normal denucleation (4 L), followed by repeated degreasing twice. Suspended by adding 3 L of water to the lower aqueous methanol solution layer, 3 L of trichloromethane was added thereto. Fractionated. Subsequently, 3 L of ethyl acetate and 1 L of normal-butanol were sequentially added to the upper layer, followed by fractionation. Anhydrous Na ₂ SO ₄ was added thereto, followed by dehydration. The mixture was decompressed and concentrated to obtain 10.5 g of dichloromethane, 4.8 g of ethyl acetate, and 20.3 of normal-butanol. g fractions were obtained respectively.

The normal-butanol fraction was boiled for 4 hours to obtain a hot water extract, and then passed through HP-20 resin (Diaion, Mitsubishi chemical industries, tokyo) to freeze-dry the unadsorbed eluate. Hereinafter, the eluate is referred to as a radish extract.

Flaming SOD1  Separation and purification

DEAE-Sepharose fast flow resin (2.5 × 14 cm; Amersharm, USA) was charged to fully equilibrate the column with a flow rate of 20 ml / min with 10 L of 20 mM Tris-HCl, pH 8.0 buffer. After applying the hot water extract to the DEAE-Sepharose column, the resin-bound protein was fractionated at a concentration of 0-350 mM NaCl, and fractionated by 70 ml while flowing a total of 10 L at a flow rate of 12 ml / min to measure SOD1 activity. The highest fractions were collected. Phenyl-Sepharose chromatography was performed on the SOD1 active fractions obtained through DEAE-Sepharose chromatography. HiPrep Phenyl-Sepharose column (16 × 10 cm; Amersharm, USA) was sufficiently equilibrated with 20 mM Tris (pH 8.0) buffer containing 1.2 M ammonium sulfate. Ammonia sulfate was added to each SOD1 fraction dialyzed with 20 mM Tris (pH 8.0) buffer to 1.2 M, loaded to the column at a flow rate of 4 ml / min and flowed back into the same buffer. In this process, the effluent was fractionated by 40 ml, and then SOD1 activity was measured and collected. SOD1 active fractions obtained through Phenyl-Sepharose chromatography were chromatographed on a Superose-12 10/300 column (Amersharm, USA). First, the column was sufficiently equilibrated with 500 ml of 20 mM Tris pH 8.0 buffer containing 100 mM NaCl. At this time, the flow rate was adjusted to 3.5 ml / min. Each sample subjected to phenyl-Sepharose chromatography was concentrated by ultrafiltration (ultrafiltration; viva spin from NOVEX), NaCl and glycerol were added to 100 mM and 7% by weight, respectively, and loaded on a column, and the above-mentioned equilibration was performed. 10 ml of each fraction was collected under the same conditions. Uno-Q1 ion exchange chromatography was performed on the fractions with high SOD1 activity obtained through Superose-12 gel filtration chromatography. After the Uno-Q1 column (BioRad, USA) was sufficiently equilibrated with 20 mM Tris pH 8.0 buffer, the dialyzed SOD1 sample was loaded at a flow rate of 2 ml / min and washed with the same buffer. After washing with 150 ml of 20 mM Tris (pH 8.0) buffer, a total of 150 ml of 0-500 mM NaCl gradient was fractionated to obtain a high-purity SOD1 final purified product.

LC - NMR ( liquid chromatography - nuclear magnetic resonance ) Way

LC- ¹ H NMR was performed on a Varian VNMRS 600 MHz NMR spectrometer ( ¹ H: 600.006 MHz; Varian, USA) connected to a Varian Pro Star HPLC system (Varian, USA) using 150 μl of triple-resonant microflow cryogenic probe. It carried out using. 1D ¹ H NMR spectra were obtained in stop-flow mode and continuous-flow mode. To perform stop-flow mode ¹ H NMR, the HPLC method was initiated at 95% D ₂ O with 0.1% HCOOH / 65% ACN and concentrated to 35% D ₂ O for 35 minutes at a flow rate of 1.0 mL / min. Gradient (run time of 45 minutes total) was UV detected at 280 nm. The mother liquor sample (50 μl) was injected into a SunFire C18 (5 μm, 4.6 × 150 mm; Waters) consisting of a reversed phase column. Standard WET1D sequences were used for pre-saturation of 1H frequency in HOD, ACN and HCOOH. Data was obtained with a 9 kHz sweep width with a acquisition time of 1.82 seconds using 33 K time domain points. A large number of scans 128-512 were used to quantify the relative concentrations of each compound in the probe flow cell. ¹ H NMR spectra were referenced to ACN resonance (1.96 ppm). Continuous-flow LC-NMR experiments were conducted under the same conditions as the stop-flow NMR described above, except that it was a flow rate of 1.0 mL / min and a total run time of 180 minutes. ¹ H NMR spectra were collected through 32 scan results obtained continuously during the elution of the chromatograph.

LC - NMR - MS ( mass spectrometry ) Way

Mass spectra were measured on a Varian 320-MS TQ mass spectrometer (Varian, USA) connected to a Varian Pro Star HPLC system operating in negative electrospray ionization mode. The HPLC method was initiated at 95% D ₂ O with 0.1% HCOOH / 5% ACN and concentration gradient up to 95% D ₂ O with 0.1% HCOOH / 65% ACN for 35 minutes (run time of 45 minutes total). A flow rate of 1.0 mL / min allowed separation at a rate of 95% for 280 nm UV detection and 5% for MS detection.

Test substance, positive control SOD , Positive control group Cafe Iksan ( caffeic acid ) And Rat Of rat homogenate  Produce

The vulgaris extract, the main test substance, was used by filtering the above-mentioned hot water extract through a 0.45 μm syringe filter. Antioxidant protein SOD1 was used to dissolve the final active concentration to 100 units / ml using pure water, and caffeic acid was dissolved in 10 mg / ml concentration in 95% ethanol. The enzyme source containing ADH and ALDH was dissolved in lyophilized S9 rat liver homogenate (MOLTOX Co., USA) in 5 mM sodium phosphate buffer (8 ml) containing 0.1% bovine serum albumin (0.4 ml). It was used after filtration with a syringe filter.

Animals and processing

Male C57BL / 6 (23-25 g) mice were purchased from DAE HAN BIOLINK CO., LTD. (Chungbuk, Korea). Animals were subjected to temperature (22 ± 2 ° C) and humidity (55 ±) with a 12 hour light / dark condition cycle (light, 7 am to 7 pm; cancer, 7 pm to 7 am) one week before the experiment. 5%)-adapted in a controlled room. Laboratory feed and tap water have been freely accessible. The developed mouse model (binge drinking) was used for the ethanol challenge (challenge). The model is a layout to obtain blood alcohol levels, behavioral effects and physiological changes comparable to those seen in human binge drinking. Mice were assigned to be treated with 0.5, 1 or 2 mg / kg of BMS (Bulberry SOD1) for 6 days prior to ethanol experiments until sacrifice. Ethanol diluted with normal salt (50%) was administered orally to mice, a total of three doses of 5 g / kg every 12 hours. Mice were sacrificed 18 hours after the final ethanol administration.

Male SD (SpragueDawley; 23-25 g) rats were purchased from DAE HAN BIOLINK CO., LTD. (Chungbuk, Korea). Animals were subjected to temperature (22 ± 2 ° C) and humidity (55 ±) with a 12 hour light / dark condition cycle (light, 7 am to 7 pm; cancer, 7 pm to 7 am) one week before the experiment. 5%)-adapted in a controlled room. The weight and condition of the animals were monitored at least twice per week. Rats were bred individually and had the same amount of control diet (47% carbohydrate in calories) or alcohol LieberDeCarli formula [36% ethanol in carbohydrates, 11% carbohydrates and 18% protein (Dyets Inc., Bethlehem, PA)] These four individual groups were fed for five weeks. Food consumption was limited to the average intake of the ethanol group that was arbitrarily fed. The diet remained refrigerated. Ethanol was mixed into the feed just before feeding. Rats were salted after 2 weeks in BMS [0.5, 1 or 2 (mg / kg / day); n = 4] every day for 3 weeks. Control animals (n = 4) received only salt.

MTT Assay

MTT assay was performed to confirm the cytotoxicity of the vulgaris extract. 50 μl of MTT (5 mg / ml in PBS) solution (Sigma) was added per well, followed by incubation at 37 ° C. for 2 hours in a 5% CO ₂ incubator, and the formazan crystals produced on the MTT-treated plate were transferred to DMSO. The absorbance was measured at 570 nm.

LDH  Enzyme Activity Measurement

LDH enzyme activity was measured according to the manufacturer's protocol using a kit purchased from Sigma or Asan Pharmaceutical (Ansan, Korea). Briefly, 1.0 ml of pyruvate substrate was placed in a NADH vial and pre-reacted at 37 ° C. for 3 minutes, and then 0.1 ml of the culture solution was added and mixed well, followed by further reaction at 37 ° C. for 30 minutes. 1.0 ml of color reagent was added to each vial for 20 minutes at room temperature, and 10 ml of 0.4 N NaOH solution was added to measure absorbance at 525 nm.

Geology Peroxidation  And GSH  Determination of the level

Liver lipid peroxidation levels were determined by measuring levels of TBA-reactive substances. Briefly, the sample was mixed with a TBA reagent comprising 0.375% TBA and 15% trichloroacetic acid dissolved in 0.25 N hydrochloric acid. The reaction mixture was reacted for 30 minutes in a boiling water bath and then centrifuged for 5 minutes at 2,000 x g. The absorbance of the supernatant was measured at 535 nm. Liver GSH levels were measured by color development using Ellman's reagent and glutathione reductase. Samples were mixed with 0.1 M sodium phosphate buffer (pH 7.5) containing 5 mM EDTA, 0.6 mM 5,5-dithiol-bis (2-nitrobenzene acid), 0.2 mM NADPH and glutathione reductase, at room temperature for 2 minutes. Reacted for a while. The absorbance of the product was measured at 412 nm. GSH content was determined by a standard curve made of known concentration of GSH.

CYP2E1  Enzyme Activity and Expression

The liver was quickly harvested, weighed and perfused with refrigerated 0.15 M KCl, followed by 0.15 M KCl, 0.1 mM EDTA, 1.0 mM dithiothreitol (DTT) and 0.01 mM phenylmethoxysulfonyl fluorine in a PotterElvehjem homogenizer (Laton Korea Co. LTD). Homogenization was performed by addition of a 4-fold dose (w / v) of 10 mM Tris-HCl, pH 7.4, including the ride. Hepatic microsome fractions were obtained via deviation centrifugation. This fraction was used to determine CYP2E1-specific oxidative activity. Aniline hydroxylase activity was determined by measuring p-aminophenol formation and microsomal protein levels were determined by Bradford method (using bovine serum albumin as standard). CYP2E1 was detected immunochemically.

Serum biochemistry

Alanine transaminase (ALT), aspartate transaminase (AST) and albumin levels were measured to assess liver damage. ALT and AST activity, or albumin levels, were measured using a diagnostic kit (Sigma Chemical Co.) using spectrophotometry. Briefly, centrifugation was performed at 10,000 × g for 10 minutes immediately after sample collection. Serum was stored in an 80 ° C. freezer until analysis. Serum ALT, AST and albumin levels were measured using an ELISA reader (Varioskan, Thermo Electron Co.).

Histological investigation

Fresh liver tissue was soaked in neutral-buffered formalin for 24 hours. The immobilized tissues were processed according to the usual standard methods, embedded in paraffin and sectioned, followed by deparaffinization and rehydration. The degree of ethanol-induced hepatitis was assessed by examining the morphological changes in liver sections by hematoxylin and eosin staining.

liver TG  Content investigation

To determine total glyceride (TG) content, 100 mg of liver was homogenized in 1 ml of 50 mM NaCL. 4 ml of chloroform: methanol (2: 1) were added to each sample. The sample was centrifuged and the organic layer was transferred to another tube and dried under nitrogen. The resulting pellet was dissolved in phosphate buffer containing 1% Triton X-100 and TG content was determined using an enzyme reaction kit (Sigma Chemical Co.).

immune Blot  analysis

Fast liver weighing and perfusion with refrigerated 0.15 M KCl followed by a fourfold dose of 0.15 M KCl, 0.1 mM EDTA, 1.0 mM dithiothreitol (DTT) and 0.01 mM phenylmethoxysulfonyl fluoride in a PotterElvehjem homogenizer Homogenized by addition of 10 mM Tris-HCl, pH 7.4. Protein concentrations in liver homogenates were determined by Bradford method (using bovine serum albumin as standard). Liver homogenates were electrophoresed on 8-10% SDS-polyacrylamide gels and transferred electrically to polyvinylidene difluoride membranes and then signals were detected using an ECL chemiluminescent system (Amersham, Buckinghamshire, UK). Representative blotting results are shown in each figure and one sample per treatment is included in the blot.

Statistical analysis

All experiments were conducted in two pairs. Mean ± standard deviations were calculated from each group and TukeyKramer test was used to assess statistical significance at confidence levels below 0.05 ( p <0.05).

Experiment result and additional discussion

Flaming  Collagen Synthesis of Extracts Inhibitory ability  Confirm

After inducing hepatic fibrosis using chemicals in the primary cultured hepatic stellate cells, the hepatic fibrosis inhibitory activity of the sample was measured, and the collagen production inhibitory activity of the BB extracts (BM extracts) was confirmed (FIG. 1). Cytotoxicity experiments of hematopoietic cells in hepatic stellate cells showed no cytotoxicity at concentrations below 200 μg / ml (no results). Thereafter, the extract was used at a concentration of 200 ㎍ / ml or less, the results of treatment of various fractions of the extracts of hepatic stellate cells isolated from primary cultured experimental animals did not show any cytotoxicity (Fig. 1).

In order to measure the amount of collagen, proline contained in a large amount of collagen was used. Radioisotope-labeled proline ( ³ H-proline) was measured and the concentration of ³ H-proline was reduced in a concentration-dependent manner when the extract was treated (FIG. 2A). In addition, the amount of hydroxyproline, a collagen precursor, was also concentration-dependently reduced with the treatment of the radish extract (FIG. 2B). More specifically, when the fraction of the extract was treated, the amount of hydroxyproline was reduced in the extract and the SOD-containing fractions (FIG. 2C). In addition, it was confirmed once again the inhibition of collagen synthesis in hepatic stellate cells through Marson-Trichrome staining (FIG. 2D) and immunostaining (FIG. 2E).

The above results indicate that the Burberry extract and its fractions inhibit the synthesis of collagen in hepatic stellate cells.

Chemical-induced Liver fibrosis  In animal models Flaming  Effect of Extract

Hepatic fibrosis leads to extracellular matrix collagen, which is activated after hepatic cell damage due to continuous alcohol absorption.In this case, matrix metalloprotease (MMP) and tissue inhibitory mataloprotease (TIMP) Promoted by increasing expression. The radish extract of the present invention was treated in a chemical-derived liver fibrotic animal model and the expression of MMP and TIMP genes involved in fibrosis promotion was measured. As a result, the synthesis of MMP-2 was inhibited in a concentration-dependent manner in the group treated with the extract of the present invention (FIG. 3A).

In addition, a physicochemical test of serum was performed to evaluate liver damage (Table 1). As can be seen in Table 1, the liver damage indicator enzyme activity (AST, ALT and hepatic lipid peroxidation) in the carbon tetrachloride-induced liver fibrosis animal model was reduced in a concentration-dependent manner.

Concentration-dependent Restorative Effect by Pretreatment of Radical Extract in Carbon Tetrachloride-Induced Liver Fibrosis Rats. process ALT (IU / L) AST (IU / L) Liver Lipid Peroxidation
(MDA; nmol / g) Control group
BM
Carbon tetrachloride (CCl ₄ )
BM (10 mg / kg) + carbon tetrachloride
BM (50 mg / kg) + carbon tetrachloride
BM (100 mg / kg) + carbon tetrachloride 334
354
2,453191
1,759162
92775
1116 662
683
3,472151
3,021103
1,12471
21145 3.210.4
3.770.4
18.60.9
14.10.7
9.20.4
5.60.4

In addition, Marson-trichrome staining (FIG. 3B) and immunostaining (FIG. 3C) confirmed that the synthesis of collagen in hepatic stellate cells was inhibited in carbon tetrachloride-induced liver fibrotic animal model.

The above results indicate that the extract of Buttercup restores liver damage in hepatic stellate cells.

Flaming  Extract Oxidative  stress Inhibitory ability

Meanwhile, the present inventors treated liver chemicals (eg, tert -butylhydroperoxide (t-BHP)) in hepatocytes and rats of primary cultured experimental animals to induce hepatotoxicity, and then measured the hepatocyte recovery by treating the radish extract of the present invention. The oxidative stress inhibitory ability was investigated. As a result, in the primary cultured hepatocytes (BM) and its fractions (BM SOD1, BMS) showed a hepatocellular protective effect against oxidative stress concentration-dependently (Fig. 4 and Table 2). In addition, as shown in the results of the physicochemical test of the serum (Table 3), hepatic damage indicator enzyme activity (AST, ALT and hepatic lipid peroxidation in the trumpet extract-treated group in the t-BHP-induced liver fibrosis animal model). ) Was concentration-dependently reduced, and glutathione levels were recovered concentration-dependently to normal levels.

Concentration-dependent effect of the Buttercup Extract Fraction (BMS) on t-BHP-induced oxidative hepatotoxicity. Treatment (mg / kg) LDH emission
(% for t-BHP) MTT
(% Of control) Liver Lipid Peroxidation
(MDA; liver nmol / g) Glutathione (mg / g liver) Control group
BMS
t-BHP
BMS (0.5 mg / kg) + t-BHP
BMS (1.0 mg / kg) + t-BHP
BMS (5.0 mg / kg) + t-BHP 334.1
384.2
10011
838.2
545.9
415.1 10012
9610
324.1
547.1
8211
9412 1.650.18
1.460.17
7.550.81
6.360.52
2.910.32
1.840.95 63.87.2
64.27.3
12.30.14
18.21.6
41.64.3
58.26.2

Effect of pretreatment of Flax Extract Fraction (BMS) on t-BHP-induced oxidative hepatotoxicity. Treatment (mg / kg) ALT (IU / L) AST (IU / L) Liver Lipid Peroxidation
(MDA; liver nmol / g) Glutathione (mg / g liver) Control group
BMS
t-BHP
BMS (0.5 mg / kg) + t-BHP
BMS (1.0 mg / kg) + t-BHP
BMS (5.0 mg / kg) + t-BHP 436
385
27531
24324
12215
617 963
934
76285
61165
32441
11412 819
789
18620
16418
12114
9410 1.820.21
1.880.20
1.230.13
1.360.16
1.740.18
1.840.19

In addition, the Burberry extract fraction reduced oxidative stress in hepatocytes in a concentration-dependent manner (FIG. 5).

The above results indicate that the Burberry extract fraction (BMS) has a protective effect against liver damage by oxidative stress.

Flaming  Alcohol Degradation of Extracts

The present inventors investigated the effect of the radish extract on blood alcohol concentration and its degradation phase in experimental animals. As a result, when the rats were orally administered with alcohol, the mussel leaching liquor increased the blood alcohol loss rate in comparison with the alcohol (FIG. 6). When alcohol was orally administered to rats, the time to peak blood alcohol level was the same for both alcohol and fire leaching liquors. Therefore, it is thought that the blood alcohol lowering effect of the vulgar leaching liquor does not affect alcohol absorption in the gastric mucosa, but increases the rate of alcohol decomposition in the liver, leading to a decrease in blood alcohol concentration.

Acute alcohol model

As can be seen in Table 4, MDA production in the ethanol-treated group increased by 3-fold compared to the control. Consistent with serum levels of ALT and AST, BMS pretreatment significantly reduced ethanol-induced hepatic lipid peroxidation (Table 4).

Effect of BMS on blood biochemistry. Processing (n = 4) ALT (IU / L) AST (IU / L) MDA (nmol / g) Control group
ethanol
Ethanol + BMS 0.5
Ethanol + BMS 1.0
Ethanol + BMS 2.0 7.800.30
14.21.80 ^#
12.21.20
11.20.50 ^*
9.601.10 ^* 21.61.20
41.54.80 ^#
43.55.50
35.53.80 ^*
26.81.80 ^* 48528
185052 ^#
148565
125048 ^*
108557 ^*

Mice were pretreated with BMS (0.5, 1.0 or 2.0 mg / kg, orally) once daily for six consecutive days. Control mice received salts. After 6 days, ethanol diluted with normal salt was administered orally to the mice three times at a dose of 5 g / kg every 12 hours. Hepatotoxicity was determined by measuring serum activity after 18 hours. Values are mean ± standard deviation for four mice. # P <0.05 significantly different from control. * P <0.05 significantly different from ethanol treated group.

liver GSH  On the level BMS Effect of:

Ethanol treated mice showed a significant decrease in liver GSH concentrations compared to control mice. Pretreatment of BMS markedly defended dose-dependently the deficiency of GSH caused by ethanol (FIG. 7).

CYP2E  For activity and protein expression BMS Effect of:

Pretreatment of BMS resulted in dose-dependent defense efficacy against ethanol-induced hepatotoxicity in mice. Using immunoblotting assays, we investigated the effect of BMS on CYP2E2 (cytochrome P450 2E2) protein expression. Liver microsomes derived from BMS-treated mice were isolated by SDS-PAGE, followed by immunoblotting with anti-CYP2E1 antibody. As can be seen in FIG. 8, ethanol administration increased CYP2E1 expression levels by 2.5-fold compared to the control 10 hours after the last dose. There was no effect on the expression of BMS uptake CYP2E1 for 6 days (no results shown). However, the increase in CYP2E1 expression in alcoholic liver was significantly inhibited by BMS dose-dependently. Therefore, we investigated the effect of BMS on hepatic microsome CYP2E1-specific monooxygenase activity. As can be seen in Figure 8, the liver microsome fraction extracted from BMS treated mice showed a surprising decrease in the hydroxylation of aniline, a CYP2E1-specific substrate. Inhibitory activity of BMS on the hepatic microsome CYP2E1-specific monooxygenase activity was also confirmed in the pyridine-induced hepatic microsomal response. The above results indicate that inhibition of CYP2E1 by BMS in mice is important for the hepatoprotective effect of BMS on ethanol.

Chronic alcohol model

After 4 weeks of chronic alcohol diet, ALT and AST activities were increased (Table 5). Increased ALT and AST activity by alcohol administration tended to decrease after BMS treatment. BMS treatment (2 mg / kg) improved ALT and AST activity to a level similar to that of the control. Chronic alcohol administration to rats markedly reduced albumin levels, which were slightly recovered by BMS (Table 5).

Effect of BMS on blood biochemistry. Processing (n = 4) ALT (IU / L) AST (IU / L) Albumin (g / dL) Control group
ethanol
Ethanol + BMS 0.5
Ethanol + BMS 1.0
Ethanol + BMS 2.0 9.800.20
21.42.50 ^#
17.81.50 ^*
15.20.50 ^*
10.41.70 ^* 22.61.70
41.54.80 ^#
33.52.50 ^*
28.53.80 ^*
23.81.80 ^* 4.40.2
3.20.3 ^#
3.80.3
3.60.3
4.10.7 ^*

Rats were reared for 5 weeks in a control or alcohol-containing diet. Two weeks after the diet, rats were treated with BMS (0.5, 1.0 or 2.0 mg / kg) daily for 3 weeks. ALT and AST activity, or albumin levels, were measured in rat plasma. Values are mean ± standard deviation for four rats. # P <0.05 significantly different from control. * P <0.05 significantly different from ethanol treated group.

liver TG  On the level BMS Effect of:

Chronic alcohol-dietized rats showed a significant increase in liver TG concentrations compared to control rats. TG measurements showed that large amounts of triglycerides accumulate in the liver of alcohol-dietized rats. According to increased liver triglycerides and histological scoring, all alcohol-dietized rats developed hepatitis. However, the onset of hepatitis was attenuated by BMS (FIG. 9). The above results indicate that the BMS diet can prevent alcoholic hepatitis. To investigate the effect of BMS on alcoholic hepatitis, we analyzed histopathologically the amount of fat deposited in the liver. If we were unable to observe the pathological changes, liver tissue was histologically normal (FIG. 10). Chronic alcohol diet for 5 weeks resulted in fat deposition and faint microvesicular and macrovesicular fat droplets were observed (FIG. 10), which resulted in degenerative morphological changes in the liver. The pathological changes of ethanol-induced liver described above were markedly suppressed in BMS-treated rats (FIG. 10C).

AMPK Phosphorylation of BMS Effect of:

AMP-activated protein kinase (AMPK) regulates fat metabolism in the liver. In addition, inhibition of AMPK by alcohol diet is associated with reduced phosphorylation of the target protein acetyl-CoA carboxylase (ACC). Chronic alcohol diet for 5 weeks significantly reduced the level of phosphorylated AMPK in liver homogenates (FIG. 11). Treatment of BMS in alcohol-dietized rats for 3 weeks resulted in the recovery of AMPK phosphorylation. Our results indicate that the expression of AMPK protein decreases with a chronic alcohol diet, which is restored by three weeks of BMS treatment.

ACC  Phosphorylation Phase BMS Effect of:

We measured the levels of phosphorylated ACC (rate-limiting enzymes involved in fatty acid synthesis in the liver). Alcohol administration inhibited ACC phosphorylation in the liver, while BMS treatment for 3 weeks improved the effect of the alcohol (FIG. 12). The results of the inventors show that the expression of phosphorylated ACC protein is reduced following a chronic alcohol diet, which is restored by 3 weeks of BMS treatment.

In Vitro ( in vitro ) ADH ( Alcohol dehydrogenase A) Promoting activity:

Determination of ADH activity was performed by Blandino's method (Bostian. Et . al . , 1978). Briefly, 5 mM sodium phosphate buffer (pH 7.85-8.0) was used as the basic reaction system. After the enzymatic reaction between ethanol and NAD ⁺ by the activity of ADH present in the enzyme source, the degree of NADH formed as a result was measured by absorbance 340 nm. Determined by measuring at The reaction system compositions of the SOD control and the caffeic acid control, as the bulge extract, negative control (BLANK) and positive control for this test reaction, are described in Table 6 below.

Effect of BMS on ADH active phase. Test Types and Components Negative control group
( BLANK ) Cafe Iksan
Control group SOD
Control group BMS Test substance - 0.3 ml
(in EtOH) 0.3 ml 0.3 ml 95% ethanol (EtOH) 0.3 ml - 0.3 ml 0.3 ml 30 mM NAD ⁺
(Concentration in reaction buffer) 0.4 ml 0.4 ml 0.4 ml 0.4 ml Enzyme
(Rat homogenates in reaction buffer containing 0.1% BSA) 0.4 ml 0.4 ml 0.4 ml 0.4 ml Reaction buffer
(5 mM sodium phosphate buffer) Final dose, 3 ml

As described above, after the reaction system was configured, the absorbance of the reaction system was stabilized by pre-incubation for 30 to 5 minutes. Then, after measuring the absorbance at 340 nm and reacted for an additional 5 minutes, it was analyzed by measuring the change in absorbance at 340 nm. The effect on the ADH activity of the sample was expressed based on the negative control group (BLANK) without addition of the sample, in terms of the degree of absorbance in which the treatment of the sample was increased relative to the reference (% relative activity).

In vitro ADH experiment is an enzyme activity experiment in which the enzyme reaction of ethanol and NAD + proceeds by the ADH enzyme activity of a rat liver homogen present in vitro, and the degree of generation of NADH formed from the product is compared. Negative control without sample addition to the reaction system resulted in an NADH absorbance increase of 8.8 ± 0.89% through enzymatic reaction for 5 minutes, and an NADH absorbance increase of 9.48 ± 5.97% for 100 unit / ml SOD treatment group. , 1 mg / ml of the caffeic acid treated group resulted in an NADH absorbance increase of 12.08 ± 4.16%. In contrast, the treatment group of the extract of the present invention (1 mg / ml) induced an increase in absorbance of 18.61 ± 6.76%. Therefore, compared to the negative control group, only the treatment group of the radish extract showed a statistically significant ADH activity increase effect (Fig. 13).

In bito ALDH ( aldehyde dehydrogenase A) Promoting activity:

The ALDH activity of the vulgaris extract was determined by Bostian's method (Bostian. Et . al . , 1978). Briefly, 5 mM sodium phosphate buffer (pH 7.85-8.0) is used as a basic reaction system. After the enzymatic reaction between acetaldehyde and NAD + by the activity of ALDH present in the enzyme source, the degree of NADH formed as a result is measured. Absorbance was confirmed by measurement at 340 nm. The reaction system composition of the SOD control and the caffeic acid control as a bulge extract, a negative control group (BLANK) and a positive control group for this experimental reaction is described in Table 7 below.

Effect of BMS on ALDH active phase. Test Types and Components Negative control group
( BLANK ) Cafe Iksan
Control group SOD
Control group BMS Test substance - 0.3 ml 0.3 ml 0.3 ml 0.5 M acetaldehyde 0.3 ml 0.3 ml 0.3 ml 0.3 ml 30 mM NAD +
(Concentration in reaction buffer) 0.4 ml 0.4 ml 0.4 ml 0.4 ml 0.33 M 2-mercaptoethanol 0.1 ml 0.1 ml 0.1 ml 0.1 ml Enzyme
(Rat homogenates in reaction buffer containing 0.1% BSA) 0.4 ml 0.4 ml 0.4 ml 0.4 ml Reaction buffer
(5 mM sodium phosphate buffer) Final dose, 3 ml

After the reaction system was configured as described above, the absorbance of the reaction system was stabilized by pre-treatment for 30 to 5 minutes. Then, the absorbance was measured at 340 nm and reacted for an additional 5 minutes, and then analyzed by measuring the change in absorbance at 340 nm. The effect on the ALDH activity of the sample was expressed on the basis of the negative control group (BLANK) without addition of the sample, in terms of absorbance increased relative to the reference (% relative activity).

In vitro ALDH experiment is an enzyme experiment in which the enzyme reaction of acetaldehyde and NAD + proceeds by the ALDH enzyme activity of rat liver homogens present in vitro, and the degree of generation of NADH formed from the product is compared. Negative control (BLANK) without sample addition to the reaction system resulted in an increase in NADH absorbance of 5.1 ± 2.46% through enzymatic reaction for 5 minutes, and 6.48 ± 2.34% of NADH for 100 unit / ml SOD treatment group. An increase in absorbance resulted in an NADH absorbance increase of 9.84 ± 1.26% for the 1 mg / ml caffeic acid treated group. In contrast, the treatment group of the extract of the present invention (1 mg / ml) resulted in an increase in absorbance of 11.46 ± 1.67%. In other words, the caffeic acid treatment group and the vulgaris extract treatment group showed a statistically significant increase in ALDH activity compared to the negative control group (Figure 14). In addition, the group of foliar extract showed about 1.2 times higher activity than the same amount of caffeic acid treated group (Figure 14).

Flaming  Fermented extract preparation and Flaming  Hangover improvement active ingredient separation / purification / structure analysis

The normal-butanol fraction was loaded on a Sephadex LH-20 column (GE Healthcare) and eluted with methanol to obtain a fraction showing ADH enzyme activity (ie, hangover improvement activity). Subsequently, the hangover improvement active fraction was subjected to silica gel chromatography (Merck), and then a fraction showing hangover improvement activity was obtained using a mixture of chloroform and methanol as a developing solvent. Finally, the active ingredient was separated, identified and analyzed using HPLC-Profiling / LC-NMR / LC-UV-MS method as shown below (FIG. 15).

Active profile

n-BuOH extract aliquots were fractionated time-based using semi-preparative HPLC at the following conditions: (a) initiation at 88% H ₂ O with 0.1% HCOOH / 12% MeCN; (b) concentration gradient (for 40 minutes) at a flow rate of 4.0 mL / min to 55% H ₂ O with 0.1% HCOOH / 45% MeCN; And (c) UV detection at 280 nm using Waters SunFire C18 (19 150 mm, 5) column. A total of 25 fractions were collected and concentrated via vacuo (FIG. 16). As a result, we isolated / identified a total of six compounds. The six compounds include scopoletin, ferulic acid, isoferulic acid, chlorogenic acid, gallic acid and cimicifugic acid. The ¹ H NMR and the structure thereof were identified as follows (Fig. 16):

Scopoletin: ¹ H-NMR (on-line, 600 MHz recorded in ACN-D ₂ O eluate); 6.20 (1H, d, J = 9.4 Hz, H-3), 7.89 (1H, d, J = 9.4 Hz, H-4), 7.11 (1H, s, H-5), 6.76 (1H, s, H -8), 3.90 (1H, s, 6-OCH ₃ ); EI-MS m / z : 192 [M + H] (C ₁₀ H ₈ O ₄ ).

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(Peak 1)

Ferulic acid: UV _max 242, 276, 294, 324 nm; ¹ H NMR (on-line, 600 MHz recorded in ACN-D ₂ O eluate); 4.14 (3H, s, 3-OCH ₃ ), 7.12 (1H, d, J = 18.9 Hz, H-), 7.93 (1H, d, J = 9.6 Hz, H-5), 8.11 (1H, brd, J = 9.6 Hz, H-6), 8.10 (1H, brs, H-2), 8.64 (1H, d, J = 18.9 Hz, H-) (C ₁₀ H ₁₀ O ₄ ).

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(Peak 2)

Chlorogenic acid: ¹ H-NMR (on-line, 600 MHz recorded in ACN-D ₂ O eluate); 1.78-2.06 (4H, m, H-2, 6), 3.60-3.61 (1H, m, H-5), 3.95-3.99 (1H, m, H-4), 4.78 (1H, bs, OH), 5.04 (1H, m, H-3), 5.64 (1H, bs, OH), 6.18 (1H, d, J = 16.0 Hz, H-8 '), 6.80 (1H, d, J = 2.0 Hz, H- 5 '), 6.98 (1H, dd, J = 2.0, 8.0 Hz, H-6'), 7.07 (1H, d, J = 2.0 Hz, H-2 '), 7.44 (1H, d, J = 16.0 Hz , H-7 '), 9.26 (1H, bs, OH), 9.68 (1H, bs, OH), 12.4 (1H, bs, OH); EI-MS m / z : 135 (C ₁₆ H ₁₈ O ₉ ).

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(Peak 3)

Gallic acid : ¹ H-NMR (on-line, 600 MHz recorded in ACN-D ₂ O eluate); 7.08 (1 H, s, H-2), 7.08 (1 H, s, H-6); EI-MS m / z : 170 [M + H] (C ₇ H ₆ O ₅ ).

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(Peak 4)

Simimifujic acid E: UV _max 230, 244, 296, 326 nm; ¹ H NMR (on-line, 600 MHz recorded in ACN-D ₂ O eluate) 3.11 (1H, d, J = 16.5 Hz, H-4), 3.22 (1H, d, J = 16.5 Hz, H-4 ), 4.15 (3H, s, 3 "'-OCH ₃ ), 6.22 (1H, s, H-2), 7.40 (1H, d, J = 19.2 Hz, H-2"), 7.61 (2H, d, J = 10.5 Hz, H-3 ', 5'), 7.81 (2H, d, J = 10.5 Hz, H-2 ', 6'), 8.02 (1H, d, J = 9.9 Hz, H-5 "' ), 8.16 (1H, brd, J = 9.9 Hz, H-6 "'), 8.28 (1H, brs, H-2"'), 8.89 (1H, d, J = 19.2 Hz, H-3 "); ESI-MS m / z 432 [M + H] (C ₂₁ H ₂₀ O ₁₀ ).

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(Peak 5)

Isoferulic acid: UV _max 216, 238, 296, 323 nm; ¹ H-NMR (on-line, 600 MHz recorded in ACN-D ₂ O eluate); 7.54 (1H, d, J = 15.5 Hz, H-7), 7.07 (1H, d, J = 2.0 Hz, H-2), 7.04 (1H, dd, J = 8.3, 2.0 Hz, H-6), 6.94 (1H, d, J = 8.3 Hz, H-5), 6.29 (1H, d, J = 15.5 Hz, H-8), 3.89 (3H, s, 4-OCH ₃ ); EIMS m / z : 193 [M + H] (C ₁₀ H ₁₀ O ₄ ).

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Flaming  From fermented extract Identified  Invitro of the compound ADH  Activity promoting effect

In vitro ADH testing is an enzyme test that compares the degree of production of NADH formed from the product by the enzymatic reaction between ethanol and NAD ^{+ due} to the ADH enzyme activity of the rat liver homogens present in vitro. The relative NADH absorbance increase rate of each test group is shown in Table 8 below.

Effect of Active Ingredients on ADH Active Phases order Treated group NADH  Absorbance increase (activity,%) One No treatment 6.4 1.24% 2 1,000 unit / ml SOD 19.5 3.4% 3 1 mg / ml caffeic acid 13.4 2.1% 4 1 mg / ml Flaky Fermented Extract 21.1 3.5% 5 1 mg / ml simicifujic acid E 10.5 2.3% 6 1 mg / ml chlorogenic acid 22.0 1.2% 7 1 mg / ml ferulic acid 15.4 1.05% 8 1 mg / ml isoferulic acid 16.4 2.5% 9 1 mg / ml gallic acid 18.0 4.2% 10 1 mg / ml scopoline 7.2 3.4%

As can be seen in Table 8, 19.5 ± 3.4% (approximately 3.05 times) for the 1000 unit / ml SOD treatment group and 21.1 ± 3.5% for the Burberry extract compared to the untreated group (6.4 ± 1.24%). 3.30 times), 22.0 ± 1.2% (about 3.44 times) for chlorogenic acid, 16.4 ± 2.5% (about 2.56 times) for isoferulic acid, and 18.0 ± 4.2% (about 2.81) for gallic acid NADH absorbance increased). Thus, the compound showed a statistically significant increase in ADH activity compared to the negative control (Fig. 17).

Flaming  From fermented extract Identified  Invitro of the compound ALDH  Activity promoting effect

In vitro ALDH testing is an enzyme test that compares the degree of production of NADH formed from the product of an enzyme reaction between acetaldehyde and NAD ⁺ by the ALDH enzyme activity of a rat liver homogenate present in vitro. The relative increase in NADH absorbance of each test group is shown in Table 9 below.

Effect of ALDH active phase active ingredient. order Treated group NADH  Absorbance increase (activity,%) One No treatment 5.4 2.3% 2 1,000 unit / ml SOD 13.4 1.2% 3 1 mg / ml caffeic acid 16.4 5.4% 4 1 mg / ml Flaky Fermented Extract 17.4 3.2% 5 1 mg / ml simicifujic acid E 4.7 1.2% 6 1 mg / ml chlorogenic acid 16.5 2.3% 7 1 mg / ml ferulic acid 11.4 1.5% 8 1 mg / ml isoferulic acid 18.4 2.3% 9 1 mg / ml gallic acid 16.4 5.1% 10 1 mg / ml scopoline 7.5 3.4%

As can be seen in Table 9, 13.4 ± 1.2% (approximately 2.48 times) for SOD-treated groups of 1,000 units / ml compared to untreated (5.4 ± 2.3%), and 17.4 ± 3.2% for unfermented fermented extracts. (Approximately 3.22 times), 16.5 ± 2.3% (about 3.06 times) for chlorogenic acid, 18.4 ± 2.3% (about 3.41 times) for isoferulic acid, and 16.4 ± 5.1% (about gallic acid) 3.04 fold) increased NADH absorbance. Thus, the compound showed a statistically significant increase in ADH activity compared to the negative control (Fig. 18).

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

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Claims (18)

A pharmaceutical composition for the prevention or treatment of alcoholic liver disorders (ALDs), which comprises a superoxide dismutase 1 (BOD) purified product (BMS) as an active ingredient, wherein the SOD1 purified product is methanol, n from a fermented fermented extract. Pharmaceutical composition, characterized in that the fluorine SOD1 purified product obtained by sequentially performing column chromatography, gel filtration chromatography and ion exchange chromatography using the hexane (n-hexane) and the fluoride extract obtained through hot water extraction.
The composition of claim 1, wherein the composition has an alcohol resolution.
The composition of claim 1, wherein the composition inhibits collagen production.
The composition of claim 1, wherein the composition reduces the expression of matrix metalloprotease (Matirix mataloprotease, MMP).
The composition of claim 1, wherein the composition has a hepatocyte protective effect against oxidative stress.
The composition of claim 1, wherein the BMS reduces alcohol-induced hepatic lipid peroxidation.
The composition of claim 1, wherein the BMS reduces the level of alcohol-induced hepatic glutathione (GSH).
2. The composition of claim 1, wherein the BMS reduces alcohol-induced liver triglycerides levels.
The composition of claim 1, wherein the BMS increases the expression of CYP2E2 (cytochrome P450 2E2).
The composition of claim 1, wherein the BMS increases phosphorylation of AMPK (AMP-activated protein kinase).
The composition of claim 1, wherein the BMS reduces the level of phosphorylated acetyl-CoA carboxylase (ACC).
The composition of claim 1, wherein the BMS is administered at a concentration of 0.5-2.0 mg / kg.
The composition of claim 1, wherein the administration of the composition is oral administration.
The composition of claim 1, wherein the active ingredient increases the activity of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH).
delete The composition of claim 1, wherein the alcoholic liver disease is hepatic steatosis, alcoholic hepatitis, liver fibrosis, cirrhosis or hepatic encephalopathy.
delete A food composition for improving a hangover comprising a bulge SOD1 purified product (BMS) as an active ingredient, wherein the bulge SOD1 purified product is obtained by extracting methanol, n-hexane and hot water from a bulberry fermentation extract. The food composition, characterized in that the fluoride SOD1 purified product obtained by sequentially performing column chromatography, gel filtration chromatography and ion exchange chromatography.

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