KR101547246B1 - Composition for prevention or treatment of liver disease comprising Vigna nakashimae extract - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of liver disease, wherein the moxibustion extract, which is an effective ingredient of the present invention, has remarkable effects on the improvement of insulin resistance by inhibiting inflammation of the liver and fat accumulation in hepatocytes, And thus can be effectively used as a preventive or therapeutic agent for liver diseases due to the activity of alleviating stress of the endoplasmic reticulum.

Description

[0001] The present invention relates to a pharmaceutical composition for prevention or treatment of liver diseases,

The present invention relates to a pharmaceutical composition for the prevention or treatment of liver disease, wherein the moxibustion extract, which is an effective ingredient of the present invention, has remarkable effects on the improvement of insulin resistance by inhibiting inflammation of the liver and fat accumulation in hepatocytes, And thus can be effectively used as a preventive or therapeutic agent for liver diseases due to the activity of alleviating stress of the endoplasmic reticulum.

The life expectancy has been lengthened due to the dietary life and the life change due to the rapid economic development of modern society, while the metabolic diseases such as obesity are increasing.

In general, obesity is a state in which body fat is accumulated more than necessary, and the consumed remaining amount of calories consumed is converted into fat, which accumulates in various parts of the body, especially in the subcutaneous tissue and the abdominal cavity. Obesity can be formed as an adult, but most of the childhood obesity or overweight in the formation of dietary habits continue to be adults. In recent years, as the economic development of Korea has facilitated lifestyle and westernized eating habits, the consumption of animal foods increases, and as the intake of fat increases, the weight gain or obesity gradually increases. Obesity is a cause of many degenerative diseases such as diabetes, hyperlipidemia, hypertension or arteriosclerosis.

On the other hand, the present invention provides a method for producing a fat-soluble food product of a water-extract of fresh-necked (scrap), cornstarch, cornstarch, .

In addition, the category of liver disease varies from mild reversible fatty liver to progressive chronic liver disease and can lead to life-threatening conditions such as liver cirrhosis, liver failure and liver cancer. The main causes of chronic liver disease are hepatitis B virus or hepatitis C virus, excessive alcohol consumption and non-alcoholic fatty liver disease (NAFLD).

Among these, non-alcoholic fatty liver disease (NAFLD) refers to a wide range of liver injuries, ranging from simple steatosis to fatty liver disease, cholestasis, antifungal fibrosis and cirrhosis. Fatty liver disease (non-alcoholic fatty liver disease) is only indicated in stages within NAFLD (Anguilo P., 2002, N Engl. J. Med., 346: 1221-1231). Pathological features are similar to pathological features of alcohol-induced liver injury, but this only occurs in patients who do not abuse alcohol. NAFLD should be distinguished from hepatitis caused by secondary causes or from non-hepatitis caused by hepatitis, because these conditions have distinct pathogens and results. The secondary causes of fatty liver disease (lipopathy) include nutrition (e.g., protein-calorie malnutrition, starvation, total parenteral nutrition, rapid weight loss, gastrointestinal surgery due to obesity), drugs (such as glucocorticoids, synthetic estrogen, aspirin, (Eg, lipid disorders, beta lipid protein disorders, Weber-Cristian disease, galactosemia, glycogen storage), metabolic syndrome, diabetes mellitus, Diabetes, obesity, or hyperlipidemia) (Anguilo P., 2002, N. Engl. J. Med., 346: 1221-1231; MacSween RNM et al., 2002, Pathology of the Liver. Fourth Edition, Churchill Livingstone, Elsevier Science).

However, even though the incidence of such chronic liver diseases is increasing, there is no effective preventive or therapeutic method for most of the diseases of this class.

On the other hand, the crop of Vigna sp. Is a wild or cultivated plant in the mountainous country of Korea. Vigna angularis, Vigna radiata, Vigna umbellata, Vigna nakashimae, and Vigna angularis var. Nipponensis are the most common crops of red beans. In particular, red bean (Vigna angularis) has been known to be effective for kidney disease, suicide, tumor, birth defects, and is also used as a folk remedy. Saponins of red bean (DDMP-saponins) exhibit antioxidative and free radical scavenging activity, and hydrothermal extracts are known to inhibit hair whitening by stimulating tyrosinase activity. And Vigna radiata has been used for treatment of fever, detoxification, swelling, swelling and rheumatism for a long time. Skin beauty products using vitexin and isovitexin in mung bean have been developed and sold . As such, the Vigna genus is a genetically inherited crop of various physiologically active substances, scientifically proving various physiological activities, and is worthy of agricultural and industrial use.

Obesity and nonalcoholic fatty liver disease (NAFLD) are closely related to each other. Obesity-related nonalcoholic fatty liver disease (NAFLD) is known to be associated with hepatocyte inflammation and ER stress. Recently, Vignanakashimae has been known to have anti-diabetic and anti-obesity effects.

However, the precise mechanism of defense against inflammation and endoplasmic reticulum stress was not elucidated, and there was a problem in studying inflammatory liver disease caused by liver disease and endoplasmic reticulum stress.

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention The present invention has been made in order to solve the above problems, and it is a first object of the present invention to provide a method for inhibiting liver inflammation and fat accumulation in hepatocytes, prevention of hepatic diseases having excellent effect of mitochondrial stress- And to provide a therapeutic pharmaceutical composition.

A second problem to be solved by the present invention is to provide a health functional food composition for preventing or ameliorating liver disease which is excellent in the effect of inhibiting hepatic inflammation and lipid accumulation in hepatocytes, and inhibiting hepatocyte apoptosis.

In order to achieve the first object, the present invention provides a pharmaceutical composition for the prevention or treatment of liver disease, which comprises an extract of Aspergillus oryzae as an active ingredient.

According to a preferred embodiment of the present invention, the liver disease may be an inflammatory liver disease caused by endoplasmic reticulum stress.

According to another preferred embodiment of the present invention, the liver disease can be induced by at least one selected from obesity-induced non-alcoholic fatty liver disease and insulin resistance.

According to another preferred embodiment of the present invention, the prevention or treatment of the liver disease is performed by reducing the protein expression of genes including at least one selected from HMGB1 and RAGE, inflammation-related factors, .

According to another preferred embodiment of the present invention, the secretion of inflammatory cytokines may be suppressed to improve insulin resistance.

According to another preferred embodiment of the present invention, the expression of liver function enzymes may be regulated to improve insulin resistance.

According to another preferred embodiment of the present invention, an increase in protein expression of the glucose transporter-4 (GLUT-4) gene and a decrease in fatty acid (ACC) synthesis inhibits fat accumulation in hepatocytes to improve insulin resistance Lt; / RTI >

According to another preferred embodiment of the present invention, the expression of active transcription factor 4 (ATF4) and the caspase-3 gene may be decreased, thereby inhibiting hepatocyte apoptosis and alleviating the stress of the endoplasmic reticulum.

According to another preferred embodiment of the present invention, it may further comprise a pharmaceutically acceptable carrier.

In order to achieve the second object, the present invention provides a food composition for preventing or ameliorating liver diseases, which comprises an extract of Aspergillus oryzae as an active ingredient.

The present invention has a remarkable effect on the improvement of insulin resistance by suppressing hepatic inflammation and accumulation of fat in hepatocytes, and is excellent in the effect of inhibiting hepatocyte apoptosis, and thus can be effectively used as a preventive or therapeutic agent for liver diseases due to the activity of alleviating stress of the endoplasmic reticulum.

FIG. 1 is a graph showing the effect of a mugwort extract on body weight change according to ingestion of high fat diet (ND: standard diet diet group, HFD: high fat diet group, HFD + VN: Extracts were administered together)
FIG. 2 is a graph showing the effect of a mugwort extract on blood glucose changes due to high-fat diet intake (ND: general standard diet group, HFD: high fat diet group, HFD + VN: Extracts were administered together)
FIG. 3 is a graph showing the results of the effect of the mugwort extract on the sugar load according to the ingestion of high fat diet (ND: standard diet, HFD: high fat diet, HFD + VN: high fat diet, .
FIG. 4 is a graph showing the effect of the mugwort extract on the insulin load according to the high-fat diet method (ND: general standard diet group, HFD: high fat diet group, HFD + VN: Extracts were administered together)
FIG. 5 is a photograph of a micrograph of the effect of a low-fat extract on lipid accumulation in hepatocytes induced by high fat diet by normal tissue staining (H & E) and oil-red o staining (ND: : High - fat diet group, HFD + VN: high fat diet group,
FIG. 6 is a graph showing the liver TG levels measured by hepatic triglyceride colorimetric assay for the effect of the mugwort extract on fat accumulation in hepatocytes by high fat diet ingestion (ND: HFD: high fat diet group, HFD + VN: high fat diet group,
FIG. 7 is a graph and a graph showing Western blotting of the expression of GLUT4 gene showing the effect of a mugwort extract on lipid accumulation in hepatocytes caused by ingestion of a high fat diet. (ND: General standard diet group, HFD: , HFD + VN (high fat diet), and some mugwort extracts.
FIG. 8 is a graph showing the results of Western blotting for the synthesis of fatty acid (ACC) showing the effect of a mugwort extract on lipid accumulation in hepatocytes caused by ingestion of a high fat diet system (ND: General standard diet group, HFD: High fat diet, HFD + VN: high fat diet,
FIG. 9 is a graph showing Western blotting of protein expression of HMGB1 and RAGE genes showing the effect of a mugwort extract on inhibition of liver inflammation according to ingestion of high fat diet (ND: standard diet, HFD : High - fat diet group, HFD + VN: high fat diet group,
FIG. 10 is a micrograph of a TUNEL analysis showing the effect of a mugwort extract on the inhibition of hepatocyte apoptosis by ingestion of a high fat diet. (ND: General standard diet administration group, HFD: High fat diet administration group, HFD + VN: This study was carried out to evaluate the effect of dietary supplementation of ganoderma lucidum extract
11 shows the result of western blotting for the expression of caspase-3 gene protein, which shows the effect of a mugwort extract on the inhibition of hepatocyte apoptosis by high-fat diet. (ND: General standard diet group, HFD: , HFD + VN: high-fat diet and some mugwort extracts)
FIG. 12 is a graph showing Western blotting of expression of caspase-3 gene expressing the effect of a mugwort extract on the inhibition of hepatocyte apoptosis upon ingestion of a high fat diet. (ND: General standard diet group, HFD: , HFD + VN (high fat diet), and some mugwort extracts.
FIG. 13 is a graph and a graph showing Western blotting of protein expression of the active transcription factor 4 (ATF4) gene showing the effect of a mugwort extract on inhibition of hepatocyte apoptosis upon ingestion of a high fat diet. HFD: high fat diet group, HFD + VN: high fat diet group,

Hereinafter, the present invention will be described in more detail.

As described above, non-alcoholic fatty liver disease (NAFLD) caused by obesity is known to be related to inflammation of the hepatocyte and ER stress. Recently, it has been reported that Vignanakashimae has anti-diabetic effect and anti-obesity effect ought. However, the precise mechanism of defense against inflammation and ER stress was not disclosed, and there was a problem in studying liver diseases and stress related diseases of the endoplasmic reticulum.

Thus, according to a preferred embodiment of the present invention, a pharmaceutical composition for preventing or treating liver disease comprising an extract of Aspergillus oryzae as an active ingredient is provided, and the above-mentioned problems are solved. Thus, it has a remarkable effect on the improvement of insulin resistance by inhibiting hepatic inflammation and lipid accumulation in hepatocytes, and is excellent in the effect of inhibiting hepatocyte apoptosis, and thus can be effectively used as a preventive or therapeutic agent for liver diseases due to the activity of alleviating stress of the endoplasmic reticulum.

The term "obesity" in the present invention is a biological phenomenon caused by interaction of complex factors such as genetic, metabolic, environmental, and behavioral factors, and is generally recognized as excess weight. 30 or more (≒ 30% or more of the standard body weight) or a BMI of 27 or more and is associated with other circulatory diseases. The cause of obesity is known to be more than 70% of genetic predisposition, and it is known that the intake of insulin and exercise is insufficient for other environmental factors. However, recently, the imbalance between the amount of energy consumed and the amount of energy consumed is important It is also reported to be a cause. The obesity diseases of the present invention include obesity diseases known in the art that are caused by complex factors such as genetic, metabolic, environmental, and behavioral, and preferably include obesity, lipid disorders, dyslipidemia, hyperlipidemia, Triglyceride, hypercholesterolemia, low HDL levels, high LDL levels, atherosclerosis and sequelae thereof, and vascular restenosis.

The term "preventive" in the present invention refers to all actions that inhibit liver inflammation, inhibit fat accumulation in hepatocytes and inhibit hepatocyte death and inhibit or delay the onset of the disease by using a composition comprising a myrtle extract as an active ingredient It says.

The term "treatment" in the present invention refers to the use of a composition comprising an acarina extract as an active ingredient to inhibit liver inflammation, inhibition of fat accumulation in hepatocytes and hepatocyte apoptosis, Or to be benefited.

The present invention includes an extract of the mung bean as an active ingredient.

In the present invention, the term "myrtle bean seedlings" is a vine-bearing annual seedlings with beans, the leaves are alternate with three leaves, the small leaves are ovate or lanceolate and the mutation is severe. The flower is yellow, It is characterized by that there are no flowers in the flower hairs. Seeds are dark brown with spots, and I am wide, pronounced, striking and white. Among the leguminous plants native to the Korean peninsula, the wild beans close to the soybean are dolomite, and the one close to the eastern part is the eastern part of the stone. In addition, there is a characteristic that the distinction between eastern part and red bean is not clearly distinguished from that of red bean which is close to red bean. It is found mostly in Jeju Island and Jindo, and is often found on the bank of the desert near Seongsan and Taean in Chungcheongnam-do.

The mugwort extract may be obtained by extracting the mugwort extract alone or in a mixed solvent thereof using at least _one of water and a lower alcohol of C ₁ -C ₄ as a solvent.

It was confirmed that the present invention can be used as a composition for the prevention or treatment of liver disease using a mugwort extract.

The liver disease may be an inflammatory liver disease caused by ER stress, and may be caused by at least one selected from non-alcoholic fatty liver disease and insulin resistance caused by obesity.

Specifically, the composition of the present invention can improve the body weight and fasting blood glucose of the ingestion group.

As shown in Experimental Example 1, it was confirmed that the composition significantly decreased body weight and fasting blood glucose in the high-fat diet group.

As shown in FIG. 1, the body weight of the high fat diet group was significantly increased from 4 weeks, and it was confirmed that the body weight was decreased by the low fat low fat diet especially at 12 weeks.

In addition, as shown in Table 1, the weight of the fat pads of the liver and epididymis of the group fed with high fat diet decreased by the garlic extract (see Table 1).

In addition, as shown in FIG. 2, the fasting blood glucose level was significantly increased in the group fed the high fat diet group compared to the group fed the normal diet after the 8th week, but it was not decreased by the low fat low fat group. However, in the last 4 weeks, it was confirmed that fasting blood glucose was significantly decreased by the mung bean extract.

As a result of this, it was found that the mung bean extract decreased the fasting blood glucose level in the rats fed the high fat diet.

The composition may have anti-inflammatory activity of hepatocytes through reduction of protein expression of genes including at least one selected from inflammation-related factors HMGB1 and RAGE.

As shown in FIG. 9, it was confirmed that the protein expression of HMGB1 and RAGE genes increased by ingestion by the high fat diet method was reduced by the mung bean extract. Thus, the mung bean extract, which is an effective ingredient of the present invention, . ≪ / RTI >

In addition, the composition can improve insulin resistance.

Specifically, in order to inhibit the secretion of inflammatory cytokines and improve insulin resistance, as shown in Experimental Example 3, in order to examine the effect of the mugwort extract on the serum cytokine and metabolic parameters by the high fat diet, The concentration of IL-1 beta and IL-6 in the serum was measured by an ELISA method. As shown in FIG. 2, the high-fat diet for 12 weeks showed insulin, leptin and cytokines IL-1beta -6) expression was increased.

In addition, the composition can improve insulin resistance by controlling the expression of liver function enzymes. As shown in Table 2, the liver function values AST and ALT were also increased. Especially, blood cholesterol, free fatty acid and lipid were increased, and these increased blood metabolic markers were decreased by administration of mugwort extract.

The composition can improve insulin resistance by inhibiting fat accumulation in hepatocytes through an increase in protein expression of the Glucose Transporter-4 (GLUT-4) gene and a decrease in fatty acid (ACC) synthesis.

Specifically, as can be seen from Experimental Example 5, it was found that the extracts of some mugwort were more likely to inhibit the protein expression of GLUT-4 gene and fatty acid (acetyl coA carboxylase) As a result of the analysis of the effect on the expression, it was confirmed that the expression of GLUT4 gene increased in the high-fat diet-fed group due to the administration of the mushroom extract (see FIG. 7) and the decrease in fatty acid (ACC) Reference).

Thus, it was confirmed that the mugwort extract of the present invention as an active ingredient of the present invention has an effect of inhibiting fat accumulation in hepatocytes and improving insulin resistance.

The composition can reduce the expression of active transcription factor 4 (ATF4) and caspase-3 gene, thereby alleviating the stress of the endoplasmic reticulum by inhibiting hepatocyte apoptosis.

Specifically, as shown in Experimental Example 7, the TUNEL assay was performed to confirm the inhibitory effect of hepatic killing in the liver of the high-fat diet-fed group by the mugwort extract, and caspase-3 gene and active transcription factor 4 (ATF4) As a result of analysis of gene expression of proteins, the number of TUNEL-positive hepatocytes increased by ingestion of high-fat diet was small in the group administered with a little red ginseng extract, and as shown in Figs. 11 and 12, The expression of the cleaved caspase-3 gene was increased by the addition of some extracts.

In addition, as shown in FIG. 13, it was confirmed that the high-fat diet suppresses the hepatocyte apoptosis through reduction of protein expression of the active transcription factor 4 (ATF4) gene, which is increased by ingestion.

Therefore, the moxibustion extract of the present invention is an active ingredient of the present invention, which suppresses the expression of caspase-3 gene and ATF4 gene, Can be useful as a preventive or therapeutic agent.

The present invention relates to a pharmaceutical composition for the prevention or treatment of liver disease, wherein an effective component of the present invention is a compound of the formula (I), which inhibits liver inflammation and fatty accumulation in hepatocytes, It has a remarkable effect on the improvement of the resistance and is excellent in the effect of inhibiting the death of hepatocyte and can be usefully used as a preventive or therapeutic agent for liver diseases due to the activity of alleviating stress of the endoplasmic reticulum.

The composition for preventing or treating liver disease of the present invention may comprise a pharmaceutically acceptable carrier. The composition comprising a pharmaceutically acceptable carrier can be of various oral or parenteral formulations. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used.

Solid formulations for oral administration include tablet pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose or lactose ), Gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have.

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the non-aqueous solvent and the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The pharmaceutical composition may be in the form of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations and suppositories It can have one formulation.

In addition, these pharmaceutical compositions are useful for administering to a subject a rosacea extract of the present invention for treating various diseases including liver disease and / or symptoms associated therewith, as described above.

The composition of the present invention is administered in a pharmaceutically effective amount.

The term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and the effective dose level will depend on the species and severity, age, sex, The time of administration, the route of administration and the rate of excretion, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts.

The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to take into account all of the above factors and administer an amount that will achieve the maximum effect in the least amount without side effects. Typical dosages of the pharmaceutical compositions of this invention are in the range of 0.001 to 100 mg / kg on an adult basis.

The route of administration of the pharmaceutical composition may be administered through any conventional route so long as it can reach the target tissue. The composition of the present invention may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, intranasally, intrapulmonarily, or rectally, though it is not intended to be limited thereto. In addition, the composition may be administered by any device capable of transferring the active agent to the target cell.

The composition of the present invention can be used alone or in combination with methods using surgery, hormone therapy, drug therapy and biological response modifiers for the prevention and treatment of liver diseases.

In one embodiment of the present invention, the reduction of protein expression of HMGB1 and RAGE genes, which are the inflammation-related factors, is inhibited by the acne extract of the present invention, and inflammation of hepatocytes is inhibited, and the increase of protein expression of GLUT- ACC) by inhibiting lipid accumulation in hepatocytes to improve insulin resistance and by inhibiting hepatocyte apoptosis through reduction of protein expression of active transcription factor 4 (ATF4) and caspase-3 gene, And can be usefully used as a preventive or therapeutic agent for diseases.

Therefore, it is possible to effectively utilize the mugwort extract of the present invention as an agent for the prevention or treatment of inflammatory liver disease caused by the stress of the endoplasmic reticulum.

According to another preferred embodiment of the present invention, there is provided a food composition for preventing or ameliorating liver disease comprising an extract of Aspergillus oryzae as an active ingredient, thereby solving the above-mentioned problems.

In case of using the mugwort extract of the present invention as a food additive, the mugwort extract may be added as it is or may be used together with other food or food ingredients, and may be appropriately used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment).

The term " health functional food " used in the present invention means a food which is prepared by using a specific ingredient as a raw material for the purpose of health assisting, or by preparing, concentrating, refining, Refers to foods that are designed and processed so that the body control function such as bio-defense, regulation of biorhythm, prevention and recovery of disease, etc. can be sufficiently exhibited to the living body by the above-mentioned ingredients. Recovery of disease, and the like.

The composition of the present invention, which contains the extract of the acanthopanax senticosus as an active ingredient, can be variously used for medicines, foods and beverages effective for prevention and improvement of liver disease. Examples of the food to which the extract of the present invention, the pharmaceutically acceptable salt thereof and the derivative thereof can be added include various foods, beverages, gums, tea, vitamin complex, health supplement foods, Powders, granules, tablets, capsules or beverages. At this time, the amount of the compound in the food or beverage may generally be from 0.01 to 15% by weight of the total food, and the health beverage composition is added in a proportion of 0.02 to 10 g, preferably 0.3 to 1 g, .

The health functional food of the present invention may contain, as an essential component, the above-mentioned nutriment extract as an essential ingredient, and a food-acceptable food supplementary additive such as various flavorings or natural carbohydrates as an additional ingredient.

Examples of the above-mentioned natural carbohydrates include conventional sugars such as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. As natural flavors other than those mentioned above, natural flavors (tautatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) .

The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the health functional food of the present invention.

In addition to the above, the health functional food of the present invention may contain flavorings such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and heavies (cheese, chocolate etc.), pectic acid and its salts, And salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like.

In addition, the health functional food of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not so critical, but is generally selected in the range of 0.0001 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

There is no limitation on the kind of health food to which the composition of the present invention can be used. In addition, the mugwort extract of the present invention can be prepared by mixing other suitable auxiliary ingredients and known additives that can be contained in health functional foods according to the selection of the person skilled in the art. Examples of foods that can be added include dairy products including meats, sausages, breads, chocolates, candies, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, Vitamin complex, and the like, and can be prepared by adding to the juice, tea, jelly, and juice prepared from the extract of the present invention as a main component.

As a result, the present invention has a remarkable effect on the improvement of insulin resistance by inhibiting hepatic inflammation and hepatic cell fat accumulation, and is excellent in the effect of inhibiting hepatocyte apoptosis, and thus can be effectively used as a preventive or therapeutic agent for liver diseases by the effect of alleviating stress of the endoplasmic reticulum.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

[ Example ]

Example  1: animal and obesity model

Male C67BL / 6J mice (3 weeks old) were purchased from KOATECH (Pyeongtaek, Korea) and reared and maintained using an animal facility of Gyeongsang National University. All experiments were performed according to the guidelines of the Laboratory Animal Room of the National Institutes of Health.

The College Animal Care Committee has approved Gyeongsang National University's Research Protocol for Animal Research (GLA-101104-M0108).

Rats were individually male / female in a 12 hour cycle, and 4-week-old rats were randomly divided into three groups (per n = 10 groups). The three groups were fed high fat diets (45%, Research Diet Inc, New Brunswick, NJ, USA) in the first group, the second diets were administered the general diet, and the last three groups were high fat diets 250 mg / kg) for 12 weeks.

Based on the previous studies, the effect of non - alcoholic fatty liver disease (NAFLD) was investigated by administering a daily dose (100 ~ 500 ㎎ / ㎏) Rats in each group were weighed three times a month for 16 weeks.

Example  2: Plant material and Some moles  Preparation of extract

(IT178464) was cultivated in Rural Development Administration of National Institute of Crop Science, Milyang, Korea in 2010.

The seedlings (1 kg) were cleaned and dried, ground in a grinder at room temperature, placed in an extraction container, and extracted with 80% aqueous ethanol (15 L) for 3 days at room temperature. The extract was lyophilized to a powder (98 g).

Example  3: Glucose tolerance test ( GTT ) And insulin resistance test ITT )

Rats were fasted for 16 hours before performing glucose tolerance test (GTT). Blood samples were collected every 30, 60, 90 and 120 minutes by intraperitoneal injection of D-glucose (2 G / kg, Sigma-Aldrich, St. Louis, Mo., USA). Blood glucose was measured using an Accu-Chek glucometer (Roche Diagnostics GmbH, Mannheim, Germany).

Insulin resistance test (ITT) was performed by administering insulin (0.75 U / kg, Hugh Mullin-R, Eli Lilly, Indianapolis, Ind., USA) to 0.1 ml of 0.9% physiological saline in rats.

Before the insulin administration and at the 15, 30, 45 and 60 minutes after insulin administration, the veins of the tail of the rats were cut and blood was taken to measure blood glucose.

Example  4: Measurement of serum metabolic parameters

For serum analysis, all mice of the three groups raised by the method of Example 1 were anesthetized with intramuscular injection of 5 mg / kg of zoletile (Virbac Laboratories, Carros, France).

In each group (n = 10), a blood sample was taken from the sinus ventricle of the rats through a 1 mL syringe through the cardiac perfusion and coagulated for 2 hours at room temperature. The blood samples were centrifuged and the serum was collected and stored at -80 ° C. until analysis.

Serum AST, ALT, free fatty acid (FFA), total cholesterol, and triglyceride (TG) levels were measured using the enzyme colorimetric assay of Green Cross Research Institute.

Serum insulin, leptin, IL-1β and IL-6 concentrations were measured using Bio-Plex Suspension Array System (Bi-Rad Laboratories, Inc., CA, USA).

Example  5: Liver triglyceride Colorimetric  analysis( Liver triglyceride colorimetric assay )

The liver tissues were excised from the three groups of rats fed with the method of Example 1, which was anesthetized in Example 3, and TG colorimetric assay kits (Cayman chemical company, Ann Arbor, MI, USA) was used to measure the concentration of hepatic triglyceride according to the manufacturer's manual.

Example  6: Preparation of tissues and samples

For tissue analysis, four rats were anesthetized with respect to the three groups raised by the method of Example 1, and heparinized saline containing 4% paraformaldehyde in 0.1 M phosphate buffer was added to each tissue And perfused with perfusion. After fixing for 6 hours, hepatic tissue was paraffin embedding and sectioned to a thickness of 5 탆.

Example  7: Oil Red ( Oil Red ) O dyeing

Oil Red O staining is commonly used to identify lipid accumulation. To confirm liver fat accumulation, liver tissue was frozen sectioned to a thickness of 5 μm, stained with 0.5% Oil Red O (Sigma), washed, stained for 45 seconds with hematoxylin (Sigma) Olympus, Tokyo, Japan).

Example  8: Western Blot

In order to extract the protein, the liver tissues frozen and stored were homogenized with a cell lysis solution.

(ACT) and cleaved caspase-3 antibody (Cell Signaling Technology, Danvers, MA, USA), glucose transporter 4 (GLUT4) antibody and HMGB1 antibody (Abcam, Cambridge, UK), ATF4 antibody and RAGE antibody ≪ / RTI > Cruz Biotechnology, CA, USA). Membranes were analyzed by the above antibody or β-actin (Sigma) and subjected to ECL (enhanced chemiluminescence substrate) (Pierce, Rockford, IL, USA).

Example  9: TUNEL ( Terminal deoxynucleotidyl transferase dUTP nick end labeling ) Method

TUNEL assays were performed according to the manufacturer's manual using in situ apoptosis kits (Roche Molecular Biochemicals, Mannheim, Germany).

Briefly, the cells were washed with phosphate buffered saline (PBS) to inhibit the activity of endogenous peroxidase, and the permeate solution (0.1% Triton X-100 in 0.1% sodium citrate) was added Lt; / RTI > A mixture containing TdT (terminal deoxynucleotidyl transferase) and fluorescence-dUTP was added to the reaction mixture, followed by reaction at 37 ° C for 60 minutes. Washed with phosphate buffer, and then observed with an optical microscope.

Example  10: Statistical analysis

The difference between the dietary group and the high fat diet group or the high fat diet group was determined by bidirectional ANOVA. Values are expressed as the mean of the mean (SEM) + - standard error. A p value <0.05 was considered statistically significant.

Experimental Example  1: High-fat diet regulates body weight and blood glucose Some moles  Effect of extract

The animal model raised by the method of Example 1 above confirmed the effect of the mugwort extract on body weight and blood sugar increased by high fat diet in a high fat dietary animal model. Body weight and fasting blood glucose were measured every 4 weeks from the start of high fat diet (45% kcal) to 12 weeks using a high fat diet animal model. The results are shown in Fig. 1 and Fig.

As shown in FIG. 1, the body weight of the high fat diet group was significantly increased from 4 weeks, and it was confirmed that the body weight was decreased by the low fat low fat diet especially at 12 weeks.

In addition, as shown in Table 1, it was found that the weight of the fat pads of the liver and epididymis in the group fed with the high fat diet decreased by the garlic extract.

In addition, as shown in FIG. 2, the fasting blood glucose level was significantly increased in the group fed the high fat diet group compared to the group fed the normal diet after the 8th week, but it was not decreased by the low fat low fat group. However, in the last 4 weeks, it was confirmed that fasting blood glucose was significantly decreased by the mung bean extract.

As a result of this, it was found that the mung bean extract decreased the fasting blood glucose level in the rats fed the high fat diet.

Experimental Example  2: High-fat diet is the most effective method for insulin resistance Some moles  Effect of extract

In obesity, insulin and glucose tolerance test were performed to confirm the anti - diabetic effect of mugwort extract. This study confirmed the effect of the extract of the mung bean on the insulin resistance improvement. The results are shown in FIG. 3 and FIG.

As shown in FIGS. 3 to 4, glucose was injected intraperitoneally on fasting, blood glucose was measured every 30 minutes, insulin was injected, blood glucose was measured every 15 minutes, and insulin load test 4), it was confirmed that the increased blood glucose level was significantly regulated by some extracts from red ginseng.

In addition, as shown in Table 2 below, it was confirmed that hyperinsulinemia and hyperleptinemia in the high fat diet-fed group were significantly decreased by the mugwort extract.

As a result, it was found that the mugwort extract of the present invention is excellent in improving insulin resistance.

Experimental Example  3: Serum cytokines and metabolism following ingestion of high fat diet On the marker  About Some moles  Effect of extract

According to the method of Example 5, the concentration of IL-1β and IL-6 in the serum was measured by ELISA assay in order to confirm the effect of the mugwort extract on the serum cytokine and metabolic parameters by the high fat diet , And the results are shown in Table 2 above.

As shown in Table 2, the 12-week high-fat diet increased the expression of insulin, leptin and cytokines (IL-1beta, IL-6) in the blood, and increased the levels of AST and ALT . Especially, blood cholesterol, free fatty acid and lipid were increased, and these increased blood metabolic markers were decreased by the administration of mugwort extract.

Experimental Example  4: Fat accumulation in the liver due to high fat diet Some moles  Effect of Extract 1

(H & E) and oil-red o staining (A) were carried out by the method of Example in order to confirm the effect of the mugwort extract on lipid accumulation in hepatocytes by the high fat diet method, TG (B) was measured. The results are shown in Fig. 5 and Fig.

As shown in FIG. 5, lipid droplets appearing in hepatocytes around the central vein were observed in the high fat diet group, but not in the low fat group.

In addition, as shown in FIG. 6, liver TG levels were measured by liver triglyceride colorimetric assay in the high fat diet group. As a result, TG levels were significantly decreased in liver TG levels due to the administration of the mugwort extract.

Experimental Example  5: Effects of high fat diet on fat accumulation in liver cells Some moles  Effect of Extract 2

The effect of the low-fat extract on the protein expression of the glucose transporter 4 gene and the expression of proteins involved in the synthesis of fatty acid (acetyl coA carboxylase) in the liver by high-fat diet was confirmed by Western blotting . The results are shown in Fig. 7 and Fig.

As shown in FIGS. 7 to 8, it was confirmed that the administration of the acacia var. Bornea extract increased the expression of the reduced GLUT4 gene in the high-fat diet group (FIG. 7) and decreased the fatty acid (ACC) 8).

In Experimental Examples 4 and 5, it was confirmed that the moxibustion extract of the present invention, which is an active ingredient of the present invention, inhibits fat accumulation in hepatocytes and has an effect of improving insulin resistance.

Experimental Example  6: Inhibition of liver inflammation by high fat diet intake Some moles  Effect of extract

The effect of some extracts of the mung bean on the protein expression of HMGB1 and RAGE genes, which are inflammation related factors, in the liver of the high - fat diet group was confirmed by Western blotting. The results are shown in Fig.

As shown in FIG. 9, it was confirmed that the protein expression of HMGB1 and RAGE genes increased by ingestion of the high fat diet was reduced by some extracts from the kidney.

As a result, it was confirmed that the mugwort extract of the present invention has an anti-inflammatory activity against hepatocytes.

Experimental Example  7: Suppression of hepatocyte apoptosis by endotracheal intubation and vesicle stress ER  stress Some moles  Effect of extract

TUNEL analysis was carried out by the method of Example 10 to confirm the effect of inhibiting hepatocyte apoptosis in the liver of the high-fat diet-fed group by the extract of the mung bean, and the protein expression of the caspase-3 gene and the activity transcription factor 4 (ATF4) gene Were confirmed by Western blotting. The results are shown in Fig. 10 to Fig.

As shown in Fig. 10, the number of TUNEL-positive hepatocytes increased by ingestion of the high-fat diet was less in the group administered with the mugwort extract.

As shown in FIGS. 11 to 12, it was confirmed that the protein expression of the cleaved caspase-3 gene, which was increased by the high-fat diet method, was decreased by the extract from the kidney.

As shown in FIG. 13, it was confirmed that the high-fat diet suppressed the hepatocyte apoptosis through reduction of the protein expression of the active transcription factor 4 (ATF4) gene, which was increased by ingestion.

Therefore, the moxibustion extract of the present invention is an active ingredient of the present invention, which suppresses the expression of caspase-3 gene and ATF4 gene, Can be useful as a preventive or therapeutic agent.

Claims (10)

The extract contains a little bittern as an active ingredient,
A pharmaceutical composition for the prophylaxis or treatment of inflammatory liver disease, which comprises an inhibitory activity of hepatocyte inflammation through reduction of protein expression of a gene comprising at least one selected from inflammation-related factors HMGB1 and RAGE. The extract contains a little bittern as an active ingredient,
A pharmaceutical composition for preventing or treating inflammatory liver disease caused by endoplasmic reticulum stress characterized by inhibiting hepatocyte apoptosis through reduction of protein expression of active transcription factor 4 (ATF4) and caspase-3 gene and alleviating endoplasmic reticulum stress. The method according to claim 1 or 2,
Wherein said pharmaceutical composition further comprises a pharmaceutically acceptable carrier. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt; The extract contains a little bittern as an active ingredient,
Prevention or treatment of nonalcoholic fatty liver disease characterized by an increase in protein expression of GLUT-4 gene and a decrease in fatty acid (ACC) synthesis, thereby inhibiting fat accumulation in hepatocytes and thereby improving insulin resistance A pharmaceutical composition. 5. The method of claim 4,
A pharmaceutical composition for preventing or treating nonalcoholic fatty liver disease, which comprises a pharmaceutically acceptable carrier. delete delete delete delete delete

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