KR101516764B1 - A composition containing Humulus japonicus extracts for preventing or treating liver disease or atherosclerosis - Google Patents

Abstract

The present invention relates to a composition for preventing or treating an insulin resistance-related disease comprising an extract of Huangshan Vine or a fraction thereof, a method of preventing or treating an insulin resistance-related disease using the composition, a method of treating an insulin resistance- A feed additive or a feed composition, and a quasi-drug composition.

Description

[0001] The present invention relates to a composition for preventing or treating liver disease or atherosclerosis,

The present invention relates to a composition for preventing or treating an insulin resistance-related disease comprising an extract of Huangshan Vine or a fraction thereof, a method of preventing or treating an insulin resistance-related disease using the composition, a method of treating an insulin resistance- A feed additive or a feed composition, and a quasi-drug composition.

Insulin is secreted from the pancreas and promotes absorption into muscle, inhibits glucose production in the liver to control blood sugar, inhibits the degradation of fatty acids in liver and adipose tissue, and stores the absorbed glucose in fat. In addition to this energy metabolism, insulin also participates in cell growth and proliferation and functions to regulate the expression of various genes. When insulin resistance (IR) is reduced due to decreased sensitivity of cells and tissues to insulin, glucose transport to the muscle cells is inhibited, glucose production in the liver increases, The increase of lipid degradation in the blood increases the inflow of fatty acids in the blood. In addition, the body recognizes that insulin is deficient, so that a greater amount of insulin is produced to induce hyperinsulinemia and the insulin resistance becomes more severe. Continued insulin resistance may lead to hyperglycemia and diabetes due to impaired glucose tolerance and may also lead to a variety of related disorders such as hyperlipidemia, hypertension, diabetes, inflammation, arteriosclerosis, heart disease, cancer or liver disease. These insulin resistance-related diseases may occur individually, but they are closely related to each other and may be accompanied by various symptoms. However, various diseases caused by such insulin resistance are not effective as therapeutic agents for other diseases, even if they are therapeutic agents for treating one kind of diseases. That is, even if the causes are the same, the therapeutic agents for the different diseases are different, so that development of a universal therapeutic agent capable of treating various diseases is required.

Insulin resistance inevitably leads to a high level of insulin in the blood and hyperinsulinemia. Hyperinsulinemia is an activity that promotes activity of sympathetic nerves or absorption of sodium in the kidney, and is a disease associated with obesity or diabetes. It is also known that hyperinsulinemia is closely related to the development of various types of cancer, such as liver cancer, breast cancer, or laryngeal cancer. Therefore, it is very necessary to develop a therapeutic agent that can reduce insulin without lowering side effects without inducing weight gain.

There are two types of diabetes, type I and type II, which are deficient in insulin or susceptible to insulin, leading to abnormalities in carbohydrate metabolism. Insulin-dependent diabetes mellitus (IDDM) is known to be the treatment of insulin-insulin-resistant diabetes mellitus until now, and insulin-dependent diabetes mellitus, that is, to treat type II diabetes Such as PPAR-γ activator, GLP-1 derivative, DPP-IV inhibitor and PTP1B inhibitor, have been developed as major diabetes medicines. However, these conventional medicines have been shown to have side effects, and thus, The development is urgent.

Obesity, a major disease associated with insulin resistance, is well known to cause chronic diseases such as fatty liver, hypertension, diabetes and cardiovascular disease. About 1.7 billion people, or about 25% of the world's population, are currently overweight (BMI> 25), and more than 300 million people in the western region, including 122,000 in the US, Europe and Japan, are classified as obese (BMI> 30). As a treatment for obesity that is marketed at home and abroad, there is "Xenical", which is the main ingredient of orlistat approved by the US FDA. In the case of Xenical inhibiting the lipase action, there is gastrointestinal side effect such as fat side, gas production and degradation of fat soluble vitamin It is known.

In addition, if the insulin resistance state continues, lipid degradation increases from the adipose tissue, and lipids such as cholesterol are increased in the blood and the arterial wall changes, resulting in hyperlipemia and arteriosclerosis.

Hyperlipidemia increases the lipid components such as blood cholesterol, the flow of blood is not smooth, and lipid components adhere to the arterial wall, causing a chronic inflammatory reaction. The narrowing of the inner wall of the artery leads to hardening of the arteries, and in the long term, thrombosis resulting from this can block the coronary arteries and cerebral blood vessels causing myocardial infarction, stroke and cerebral infarction. Since these symptoms and related diseases occur even in a non-obese state, insulin resistance may be an important factor in the development of such diseases. Currently, the drug for treating hyperlipidemia is a statin-based drug that inhibits HMG-CoA reductase, which plays an important role in the synthesis of cholesterol in the liver. Muscle toxicity and other side effects ( Toxicologic Pathology , 32 (Suppl. 2): 26-41, 2004).

In addition, atherosclerosis is a condition in which an artery becomes ruptured due to accumulation of a fatty substance (plaque) containing cholesterol, phospholipid, and calcium in the endothelium, resulting in narrowing of the elasticity and narrowing of blood supply or pressure, . Currently, a variety of statins such as HMG-CoA reductase inhibitors have been developed as therapeutic agents for arteriosclerosis. However, as with hyperlipemia, there is a need to develop more effective therapeutic agents.

Insulin resistance is also known to be a major cause of hypertension. Nitric oxide, which acts to lower blood pressure by relaxing smooth muscle of blood vessels, is required to have a smooth endothelial insulin signaling pathway during the production process. When insulin-resistant, insulin signal transduction system is disturbed and nitric oxide And the blood pressure is increased. Drugs that inhibit the binding of renin inhibitor drugs, inhibitors of angiotensin converting enzyme activity, and angiotensin and angiotensin receptors have been developed as therapeutic agents for hypertension, but there is a growing need for more effective therapeutic agents.

On the other hand, insulin resistance is known to be closely related to hepatitis and cirrhosis, which are typical liver diseases. Hepatitis can be classified into viral hepatitis and nonviral hepatitis. Viral hepatitis is caused by hepatitis virus infection such as type B or type C. Non-viral hepatitis can be classified as alcoholic hepatitis and non-alcoholic hepatitis. In general, fatty liver is often accompanied by excessive accumulation of fat over a long period of time, resulting in oxidative stress, resulting in hepatocyte damage It is thought to lead to hepatitis. In some cases, hepatitis may be caused by drugs or toxins without accompanying fatty liver. These viruses or non-viral hepatitis secrete inflammatory mediators such as tumor necrosis factor (TNF-a) and interleukin-6 (IL-6) in the process of inflammatory reaction caused by hepatocyte injury. Factors are known to disrupt the insulin signaling system and cause insulin resistance.

In the case of cirrhosis, most of the hepatocytes are destroyed, so that normal hepatocytes become insufficient and normal action of insulin on the hepatocytes can not be achieved, resulting in insulin resistance and hyperinsulinemia. And is also known to be involved in various types of cancer.

Until now, steroids or immunosuppressants have been used to treat these hepatitis, but the therapeutic effect is often limited. In addition, even in case of fatty liver, there are few medicines useful for pharmacological treatment, and exercise and diet therapy are recommended, but in fact, the treatment efficiency of fatty liver by this method is very low, and development of effective therapeutic agent is required. Therefore, it is very urgent to develop therapeutic agents such as safe hepatitis, fatty liver, liver cirrhosis and liver cancer, which are excellent in efficacy but do not cause side effects.

Under these circumstances, the present inventors have found that there is no adverse effect on insulin resistance-related diseases including hyperinsulinemia, obesity, diabetes, hyperlipidemia, arteriosclerosis, hypertension or liver disease, As a result of intense efforts to develop the substance, the extract of Hwangsam Vine lowered the triglyceride level in the blood, showed protective effect of the pancreas, decreased the levels of glutamyl oxaloacetic transaminase (GOT) and glutamyl pyruvate transaminase (GPT) The present invention has been accomplished by confirming that it is possible to inhibit the production of lipid peroxidation and to be effective for prevention and treatment in animal models of various insulin resistance-related diseases.

It is an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of insulin resistance-related diseases comprising the extract of Ornithoptera japonica or a fraction thereof.

It is another object of the present invention to provide a method for treating a disease associated with insulin resistance, comprising the step of administering a pharmaceutical composition for preventing or treating said insulin resistance-related disease to a subject in need thereof.

It is still another object of the present invention to provide a quasi-drug composition for preventing or ameliorating insulin resistance-related diseases comprising the extract of Ornithoptera japonica or a fraction thereof.

Another object of the present invention is to provide a feed additive or a feed composition for prevention or amelioration of an insulin resistance-related disease comprising the extract of Ornithoptera japonica or a fraction thereof.

It is still another object of the present invention to provide a food composition for preventing or ameliorating insulin resistance-related diseases, comprising an extract of Ornithoptera japonica or a fraction thereof.

It is still another object of the present invention to provide a method for treating an insulin resistance-related disease, comprising the step of administering the pharmaceutical composition to a subject other than a human suspected of having insulin resistance.

In order to accomplish the above object, the present invention provides, as one embodiment, a pharmaceutical composition for preventing or treating insulin resistance-related diseases comprising an extract of Ornithoptera japonica or a fraction thereof.

The term in the invention, "Humulus japonicus (Humulus japonicus "is a vine plant belonging to the genus Cannabaceae , which refers to plants that are distributed throughout Korea and throughout East Asia. The stem of the stem is made of fiber, the fruit is an antiseptic, the fruit is a diuretic However, it has not been disclosed about the use of treatment or prevention of diseases associated with insulin resistance, and the present inventors have been the first to disclose the use of the compounds of the present invention for the treatment of hyperinsulinemia, obesity, diabetes, hyperlipidemia, arteriosclerosis, hypertension, The present invention provides a method of treating or preventing an insulin resistance-related disease, which comprises administering to a human,

In the present invention, the term "Horseradish chrysanthemum extract" means an extract obtained by extracting horseradish peroxidase. Specifically, the extract may be extracted with a solvent selected from the group consisting of water, alcohols having 1 to 6 carbon atoms, and a mixed solvent thereof, and may be extracted according to a method commonly used in the art . Non-limiting examples of the extraction method include hydrothermal extraction, ultrasonic extraction, filtration, and reflux extraction. These may be performed alone or in combination with two or more methods.

In the present invention, the extract obtained by hot water extraction or cold extraction is filtered to remove suspended solid particles by filtration, for example, nylon or the like, or by freeze filtration, Spray drying and the like.

The extract of Panax ginseng is a mixture of 1 to 20 times, more specifically about 3 to 5 times the volume of water, of ₁ to 6 carbon atoms (C ₁ to C ₆ ) such as methanol, ethanol and butanol, ), Or a mixed solvent having a mixing ratio of about 1: 0.1 to 1:10, and the extraction temperature is 20 to 100 DEG C, specifically, room temperature, and the extraction period is about 2 The extract may be extracted by using an extraction method such as hot water extraction, cold extraction, reflux cooling extraction or ultrasonic extraction for 4 to 4 days. However, as an extract that can exhibit the therapeutic effect of the insulin resistance-related diseases of the present invention, But may include all of the extract, the diluted solution or concentrate of the extract, the dried product obtained by drying the extract, or the adjusted product or the purified product. The hornblende extract may be extracted from various organs of natural, hybrid, and variant plants and may be extracted from plant tissue cultures as well as from the roots, top parts, stems, leaves, flowers, It is possible.

In one embodiment of the present invention, the process of ultrasonically treating the crushed product of Panax ginseng as methanol, which is a typical alcohol, as an elution solvent, and then allowing to stand at room temperature for 15 minutes at room temperature, is repeated 10 times a day for ultrasonic extraction, followed by filtration and concentration , The sample was frozen in a cryogenic freezer and dried using a freeze dryer (Example 1). It is obvious that the same effect is obtained when extracting with alcohol of various carbon number and mixed solvent in addition to such extraction solvent.

The term "fraction " as used herein means a product obtained by performing fractionation to separate a specific component or a specific component group from a mixture containing various components.

The fractionation method for obtaining the fraction in the present invention is not particularly limited and may be carried out according to a method commonly used in the art. As a non-limiting example of the above-mentioned fractionation method, there can be mentioned a method in which a fraction obtained from the extract is treated with a predetermined solvent by treating the extract obtained by extracting the hornblende.

The kind of the solvent used for obtaining the fraction in the present invention is not particularly limited, and any solvent known in the art can be used. Non-limiting examples of the fraction solvent include polar solvents such as water and alcohol; And non-polar solvents such as hexane, ethyl acetate, chloroform, and dichloromethane. These may be used alone or in combination of two or more. When alcohol is used in the fraction solvent, C ₁ to C ₄ alcohols can be specifically used. It is apparent that the fraction of the extract of the present invention has the same effect as the extract.

The term "insulin resistance (IR) -related disease" in the present invention refers to a state in which the insulin effect is reduced due to a decrease in sensitivity of cells and tissues to insulin. The insulin resistance- Include, without limitation, diseases that can be treated or prevented. For example, one or more diseases selected from the group consisting of hyperinsulinemia, obesity, diabetes, hyperlipidemia, arteriosclerosis, hypertension and liver disease. In addition, for example, the insulin resistance-related disease is arteriosclerosis; Hyperinsulinemia; And liver disease selected from the group consisting of non-alcoholic fatty liver disease, non-fatty liver disease, end-stage fibrosis liver disease, non-viral chronic hepatitis, liver cirrhosis, acute liver injury or liver cancer.

The term "hyperinsulinemia" in the present invention refers to a state of high insulin levels in the blood, which is accompanied by obesity or diabetes, such as hyperactivity of sympathetic nervous system or absorption of sodium in the kidney.

In one embodiment of the present invention, the insulin concentration was significantly decreased in all the experimental groups treated with the horseradish peroxidase extract and the control group without the horseradish peroxidase extract, It was confirmed that there was an effect of suppressing hyperinsulinemia (Fig. 1).

The term " obesity " in the present invention is known as a representative disease associated with insulin resistance, and is diagnosed as obesity when the body mass index (BMI) is 25 or more.

In an embodiment of the present invention, when the animal model in which dietary obesity was induced by eating the high fat diet was compared with the control group in which the extract of Huangshan japonica extract was administered, the effect on obesity was confirmed, It was confirmed that the high fat diet significantly reduced weight-induced weight gain and thus had an effect of inhibiting obesity (FIG. 2).

The term "diabetes " in the present invention means a disease that occurs when insulin secretion is insufficient or insulin action and function are not sufficiently performed. In case of this disease, excessive decomposition of glycogen, protein and fat may cause liver or blood Resulting in an abnormally increased glucose concentration, resulting in diabetes and ketoneuria, resulting in an abnormality of water and electrolyte metabolism, and concomitant conditions such as circulatory disturbance, renal disorder, as well as blood concentration due to electrolyte loss. Insulin is secreted in the beta cells of the islets of the islets in the pancreas, secreted when the glucose level in the blood is increased, and secreted is suppressed when the blood glucose level is increased. The disease is divided into insulin - dependent diabetes mellitus (type I) and non - insulin dependent diabetes mellitus (type II). Diagnosis of diabetes is usually made by measuring blood glucose levels, which vary according to standards. In humans, diabetes is usually diagnosed when the blood glucose level is more than 200 mg / dl, and fasting is more than 140 mg / dl. Thus, lowering the glucose concentration in the blood or liver can treat or prevent diabetes.

In one embodiment of the present invention, the effect of improving the glucose tolerance was evaluated by administering the extract of Hwansamsun to db / db mouse, which is a representative type 2 diabetes model animal. In particular, the blood glucose level in the experimental group consumed with the Hwangsam (Fig. 3). In addition, the db / db mouse model is a model of type 2 diabetes, a model showing impaired glucose tolerance as well as insulin resistance, as well as insulin resistance. As a result, there was no difference between the control group and the experimental group in the blood glucose level before the insulin administration, but the blood glucose level started to decrease in the intake group of the horseradish peroxidase extract after 30 minutes of administration compared with the control group without the insulin. From these results, it was confirmed that the administration of the hornblende extract increased the sensitivity to insulin, thereby improving insulin resistance (FIG. 4). In addition, it was confirmed that the animal model of diabetes by streptozotocin effectively inhibited pancreatic injury and had a hypoglycemic effect due to insulin increase due to protection against pancreas (FIG. 5).

In the present invention, the term " hyperlipidemia " refers to a disease in which fat metabolism such as triglycerides and cholesterol is not properly performed and a large amount of fat is produced in the blood. Specifically, the lipid component such as triglyceride, LDL cholesterol, phospholipid and free fatty acid in blood may be increased, and may include hypercholesterolemia or hypertriglyceridemia, which occurs frequently.

In one embodiment of the present invention, it was confirmed that when an animal model in which acute hyperlipemia was induced by corn oil was orally administered a composition containing an extract of Rhodia sinensis, the effect of inhibiting the absorption of triglycerides in blood (Table 1 and FIG. 6). In particular, it showed the same or superior triglyceride inhibitory effect as Xenical, which is known as the existing therapeutic agent, and thus it was confirmed that the natural extract having no side effects is superior to the effect of suppressing hyperlipemia.

In the present invention, the term "atherosclerosis" refers to a condition in which a fatty substance (cholesterol, phospholipid, calcium, etc.) containing an oleaginous substance accumulates in the endothelium and the artery becomes hardened to lose elasticity and become narrower, And the like.

In one embodiment of the present invention, atherosclerotic effect was observed in ApoE-deficient mice, and then the effect of inhibiting arteriosclerosis by administration of Hwangsamwine extract was observed. As a result of the analysis, it was confirmed visually that red ginseng was reduced in the inner wall of the artery in the group treated with 250 mg / kg of Hansampigin extract, and it was confirmed that the fat deposition (atherosclerotic plaque) , It was confirmed that this effectively inhibited arteriosclerosis (FIG. 7).

The term "hypertension" in the present invention refers to hypertension in which the blood pressure of the artery is high and the blood pressure of the artery is high and the systolic blood pressure is 140 mmHg or more or the diastolic blood pressure is 90 mmHg or more in an adult over 18 years old.

In one embodiment of the present invention, 12-week-old male SHR rats (Spontaneously hypertensive rats), widely used as an animal model of hypertension, were fed a diet containing 500 mg / kg of hornblende extract at a concentration of 1 week, The blood pressure before and after the administration of the extract was lowered to 167 ± 8 mmHg after 1 week of administration of the hornblende compared to the systolic blood pressure of 191 ± 6 mmHg before the administration of the hornbush vine, (Fig. 8).

The term "liver disease" as used herein includes, but is not limited to, liver disease induced by insulin resistance or liver disease that can be treated by the composition of the present invention, but examples thereof include non-alcoholic fatty liver, Chronic liver disease, non-viral chronic hepatitis, liver cirrhosis, acute liver injury, liver cancer, and the like. Specifically, the composition of the present invention can exhibit liver protecting effect and lipid peroxide formation inhibitory effect.

In one embodiment of the present invention, in an animal model of acute liver damage, GOT (glutamyl oxaloacetic transaminase) and GPT (glutamyl pyruvic transaminase), which are representative indicators of liver damage in blood, , In particular, a similar effect to the silymarin administration group used as a therapeutic agent for liver disease (FIG. 10).

In addition, the degree of lipid oxidation in liver tissues produced by carbon tetrachloride was analyzed by using 4-HNE as a lipid oxidation index, and the hornblende showed a lower effect on lipid peroxide than the conventional treatment, silymarin (FIG. 11A) The lipid peroxidation in the liver tissues showed 5.8 ± 2.1 μM / mg protein in the control group, 26.7 ± 6.4 μM / mg protein in the control group and 7.9 ± 3.3 μM / mg protein in the silymarin treated group It was confirmed that the production of lipid peroxides was significantly decreased in the group to which the extract was administered (Fig. 11B).

In the present invention, the term "fatty liver" refers to a phenomenon in which abnormally deposited fat cells appear in liver cells differently from cases in which triglycerides are normal. In normal liver, about 5% is composed of adipose tissue, and triglyceride, fatty acid, phospholipid, cholesterol and cholesterol ester are the major components of fat. However, once fatty liver occurs, most of the components are replaced by triglycerides. If the liver weight is 5% or more, it is diagnosed as fatty liver. Fatty liver is caused by fat metabolism disorder in hepatocytes or defects in the process of transporting excess fat, and it is mainly caused by fat metabolism disorder in the liver. Most of the fat accumulated in the liver is triglyceride, and fatty liver can be roughly divided into non-alcoholic fatty liver due to obesity, diabetes, hyperlipidemia, drug, and alcoholic fatty liver due to excessive drinking. It is known that the above-mentioned non-alcoholic fatty liver alcohol is accompanied by fatty liver without a history, and it is known to be related to insulin resistance diseases such as obesity, diabetes and hyperlipemia.

Non-alcoholic steatohepatitis or end-stage fibrosis liver disease include not only the accumulation of fat in the liver, but also non-alcoholic fatty liver disease. Alcoholic fatty liver is caused by a high intake of alcohol, which promotes fat synthesis in the liver and does not result in normal energy metabolism. Alcohol is not stored in the body and is completely eliminated by oxidation in the liver. Specifically, the alcohol in the liver is largely divided into the alcohol dehydrogenase (ADH) pathway, the microsomal ethanol oxidizing system (MEOS ) Pathway and the catalase pathway, which are then converted to acetaldehyde, which in turn is metabolized to the acetyl salt by dehydrogenase (ALDH). At this time, acetaldehyde is toxic and can damage hepatocytes. In addition, in the pathway of micro-alcohol oxidation pathway, superoxide (O (O)) in the process of alcohol metabolism by activation of cytochrome P450-2E1 (CYP2E1; cytochrome P450 2E1) ₂ ), hydrogen peroxide (H ₂ O ₂ ), and peroxyneitrite (ROS), which are known to cause oxidative stress and cause liver damage. In addition, the metabolism of such alcohol results in the accumulation of fatty acids, resulting in the accumulation of liver fat. The fatty liver accumulated in the liver is called alcoholic fatty liver.

Fatty liver is mostly known to be associated with obesity, but it can also occur in people who are dry or normal. This finding is supported by reports that 40% (32/81) of patients with fatty liver have normal BMI ( Nucl . Med . Mol . Imaging . , 40, 243-248 (2006)). In particular, in the non-obese group, triglyceride was found to be a major factor ( J. Clin . Gastroenterol ., 40, 745-752 (2006)) and in patients with fatty liver, cholesterol and triglyceride Of the subjects were found to be significantly higher than those in the non-obese subjects.

In one embodiment of the present invention, the effect of suppressing fatty liver was observed using an animal model in which a fatty liver was induced by eating a high fat diet. In the group administered with the hornblende extract, liver triglyceride concentration was significantly lower than that of the control group, (Fig. 12). In addition, the effects of methionine - choline - deficient diets on the hepatitis animal model, which was accompanied by fatty liver, were observed. As a result, the hepatitis score obtained in the control group was 1.5 ± 0.08, And 0.5 ± 0.10 in the treated group, indicating that the extract of Panax notoginseng effectively inhibited hepatitis accompanied by fatty liver induced by methionine-choline deficient diets (FIG. 13A). The concentration of GOT and GPT, which are indicators of liver damage in blood, was also significantly lowered in the group treated with Hwangsan japonicum extract than in the control group. Thus, inhibition of infiltration of inflammatory cells in the liver inhibited hepatitis, (Fig. 13B).

In the present invention, hepatitis induced by insulin resistance may include non-fatty hepatitis not accompanied by fatty liver as well as hepatitis accompanied by fatty liver as described above.

In one embodiment of the present invention, the effect of the extract of Hwangsan japonica extract was observed by using an animal model in which hepatitis induced by fatty acids was not induced using Concanavalin A (ConA). In the experimental group treated with Hwangsamwine extract, Was significantly improved (Fig. 14A). In addition, it was confirmed that the concentration of GOT and GPT, which are indicators of liver damage in blood, was significantly decreased in the group treated with Hwangsan japonicum extract as compared with that of the control group, and thus it was confirmed that the Hwangsinchungae extract effectively reduced liver damage ).

Such hepatitis may be non-viral hepatitis and the term "non-viral" means a disease caused by an alcoholic, non-alcoholic (obesity, diabetes, hyperlipidemia) or other (toxicity, pollution, drug, , But it is not limited to the above cause and may include all hepatitis whose cause is not the virus. Specifically, the cause may be hepatitis induced by insulin resistance rather than the virus.

In addition, the liver disease of the present invention may include chronic liver disease, and the chronic liver disease may include chronic hepatitis, liver cirrhosis, liver cancer, and the like.

Cirrhosis, also called liver cirrhosis, refers to all forms of liver disease characterized by loss of significant hepatocytes, proliferation of fibrous tissue, and regenerative nodules. Liver cirrhosis can be induced by insulin resistance, and can lead to death due to complications such as ascites (boil-water symptoms), variceal bleeding, and hepatic coma.

In one embodiment of the present invention, the expression of alpha-smooth muscle actin (α-SMA) gene, which is known to be a liver cirrhosis index as compared with the control group, was 100% in the control group in the case of the liver cirrhosis-induced animal model, And the extract administration group was reduced to 58% of the control group (Figs. 15A and 15B).

In the present invention, the term "liver cancer" means a disease caused by a patient suffering from chronic liver disease caused by a liver cirrhosis patient or an alcohol. Liver disease, such as hepatitis induced by insulin resistance, and liver cirrhosis, may progress to liver cancer if it progresses or worsens further. Hepatocellular carcinoma can be diagnosed if a typical hepatic mass is seen or if the alpha-fetoprotein (AFP) is elevated in blood tests.

The cause of all of these liver diseases may vary from patient to patient, but when the liver is chronicized, the disease gradually progresses to hepatitis, liver cirrhosis, and liver cancer. Therefore, Suggesting that the levels of glutamyl oxaloacetic transaminase (GOT) and glutamyl pyruvate transaminase (GPT) may be reduced to treat or prevent liver disease.

Specifically, the composition of the present invention may have a characteristic of inhibiting the absorption of blood triglyceride and may have a blood glucose lowering characteristic due to pancreatic cell protection.

(I) a decrease in the level of glutamyl oxaloacetic transaminase (GOT), a liver damage marker in the blood; (Ii) a decrease in the level of glutamyl pyruvic transaminase (GPT), a liver damage marker in the blood; (Iii) active oxygen inhibition; (iv) reduced production of lipid oxides in liver tissue; (v) Improvement of glucose tolerance; And / or (vi) an insulin resistance improving effect.

The term "prevention ", as used herein, refers to all actions that inhibit or delay the onset of insulin resistance-related diseases such as obesity, hyperlipidemia, hypertension, arteriosclerosis, hyperinsulinemia, diabetes, liver disease, . As used herein, the term "treatment" refers to any action that improves or alleviates symptoms of the diseases upon administration of the composition of the present invention.

In a specific embodiment of the present invention, the pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier.

The composition comprising a pharmaceutically acceptable carrier may be of various forms, whether oral or parenteral. 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 tablets, pills, powders, granules, capsules, and the like, which may contain one or more excipients such as starch, calcium carbonate, sucrose or lactose lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like are also 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 esterol such as ethyl oleate.

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.

The composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount " as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will vary depending on the species and severity, age, sex, , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including co-administered drugs, and other factors well known in the medical arts.

A typical daily dose of the composition comprising the extract of the present invention or the fraction thereof as an active ingredient may be in the range of 10 to 1,000 mg / kg, specifically 10 to 500 mg / kg, Can be divided into several doses. According to a specific embodiment of the present invention, acute hyperlipemia-induced mouse model was effectively treated by oral administration once a day at a concentration of 100, 200, and 400 mg / kg, It was confirmed that the mouse model of acute liver injury induced by carbon tetrachloride can be effectively treated by oral administration once per day, by effectively treating diabetes-induced mice and orally once a day at a concentration of 500 mg / kg.

The prophylactic or therapeutic agent for insulin resistance-related diseases such as obesity, hyperinsulinemia, obesity, diabetes, hyperlipidemia, arteriosclerosis, hypertension and liver disease may be administered daily or intermittently, Alternatively, it is possible to administer two or three divided doses. When the two active ingredients are each monoglyceride, the number of administrations may be the same or different. In addition, the composition of the present invention can be used alone or in combination with other medicines for the prevention or treatment of liver disease. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art.

In another aspect of the present invention, there is provided a method for treating an insulin resistance-related disease comprising administering to a subject in need thereof a herbal extract or a fraction thereof, and / or a subject suspected of having an insulin resistance-related disease do.

In the present invention, the term "individual" means all animals, including humans, who have already developed or are capable of developing an insulin resistance-related disease, and by administering to a subject a composition comprising the extract of Panax ginseng or a fraction thereof as an active ingredient, Can be effectively prevented and treated. The term refers to whole mammals including dogs, cows, horses, rabbits, mice, rats, chickens or humans, but the mammal of the present invention is not limited by these examples. Specifically, the object may be an object other than a human.

A method for treating an insulin resistance-related disease of the present invention comprises administering a therapeutically effective amount of a horseradish extract or a fraction thereof. It will be apparent to those skilled in the art that the appropriate total daily dose may be determined by the practitioner within the scope of sound medical judgment. It can also be administered once or several times. The optimal amount and dosage interval of the individual doses of the compositions of the present invention will be determined by the nature and extent of the disease being treated, the dosage form, route and site of administration, and the age and health status of the particular patient being treated, It will be apparent to those skilled in the art that the appropriate dosage will be determined. Such dosing can be repeated as often as is appropriate. If side effects occur, dosage and frequency can be altered or decreased depending on the usual clinical practice.

The route of administration of the composition may be administered via any conventional route so long as it can reach the target tissue. The composition of the present invention may be administered intraperitoneally, intravenously, subcutaneously, intradermally, or orally, but not limited to, parenteral administration, as desired. The composition may also be administered by any device capable of transferring the active agent to the target cell.

In another specific embodiment of the present invention, the composition of the present invention may be a quasi-drug composition.

The above extracts or fractions thereof, insulin resistance-related diseases have been described above. More specifically, the composition of the present invention can be added to a quasi-drug composition for the purpose of preventing or improving insulin resistance-related diseases.

The term "improvement" in the present invention refers to the effect of alleviating the symptoms of insulin resistance-related diseases such as hyperlipidemia and diabetes by applying the quasi-drug composition comprising the extract of the present invention or fractions thereof.

The term "quasi-drug" in the present invention means a fiber, a rubber product or the like used for treating, alleviating, treating or preventing a disease of a human or an animal, Or products similar to those that are not machinery, preparations used for sterilization, insecticides and similar uses for the prevention of infectious diseases, for the purpose of diagnosing, treating, alleviating, treating, or preventing diseases of human beings or animals Means goods, machinery, or apparatus other than the apparatus, machinery or equipment of the commodity being used and used for the purpose of giving pharmacological effects to the structure and function of humans or animals. The quasi-drugs include external preparations for skin and personal hygiene products.

When the extract of the present invention is used as a quasi-drug additive, the extract may be added as it is or may be used together with other quasi-drugs or quasi-drugs, and may be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be appropriately determined depending on the purpose of use.

The external preparation for skin is not particularly limited, but may be manufactured and used in the form of an ointment, a lotion, a spray, a patch, a cream, a powder, a suspension, a gel or a gel. Such personal care products include but are not limited to soap, cosmetics, wet tissues, tissue paper, shampoo, skin creams, facial creams, toothpastes, lipsticks, perfumes, make-ups, foundations, ball touches, mascaras, eye shadows, A sunscreen lotion, a hair care product, an air freshner gel, or a cleaning gel. Further, another example of the pharmaceutical composition of the present invention is a disinfectant cleaner, a shower foam, a gagrin, a wet tissue, a detergent soap, a hand wash, a humidifier filler, a mask, an ointment agent or a filter filler.

In another aspect, the present invention provides a feed additive or a feed composition for preventing or ameliorating insulin resistance-related diseases, comprising an extract of Panax ginseng, or a fraction thereof.

The above extracts or fractions thereof, insulin resistance-related diseases are as described above.

Specifically, the extract of the present invention or its fraction may be added to a feed additive or a feed composition containing the feed additive for the purpose of preventing or improving insulin resistance-related diseases.

The term "feed additive" in the present invention means a substance added to feed for various purposes such as nutrient supplementation and weight loss prevention, promotion of digestive utilization of fibrin in feed, improvement of oil quality, prevention of reproductive disorder, improvement of conception rate, . The feed additive of the present invention corresponds to an auxiliary feed in the feed control method and is a mineral preparation such as sodium hydrogencarbonate, bentonite, magnesium oxide and complex mineral, a mineral preparation such as zinc, copper, cobalt and selenium, , Vitamins such as vitamin E, vitamin A, D, E, nicotinic acid and vitamin B complex, protective amino acids such as methionine and lysic acid, protective fatty acids such as calcium salt of fatty acid, probiotics (lactic acid bacteria), yeast culture, Fermented foods and the like, yeast preparations and the like may be further included.

As used herein, the term "feed" means any natural or artificial diet, single meal, or the like component for feeding, ingesting, digesting or suitable for the animal. Specifically, the feed comprising the composition for preventing or treating insulin resistance-related diseases according to the present invention as an active ingredient can be manufactured in various types of feed known in the art, and specifically, Feeds may be included.

Concentrated feeds include seeds containing cereals such as wheat, oats, and corn, and by-products such as rice bran, wheat bran, barley, etc., Which is the by-product of the starch residue, which is the main component of the starch residue, which is the remaining product of starch minerals, sweet potatoes, potatoes, etc., which is a byproduct obtained by concentrating the fresh liquid product obtained from fish meal, fish meal, fish residue, The animal feed such as fish soluble, meat powder, blood meal, wheat meal, skim milk powder, cheese in milk, dried whey in the case of casein production in skim milk, dry yeast, yeast, Chlorella, algae, but not limited thereto.

Roughage includes root vegetables such as wild grasses, grasses and fodder, raw turnip for feed, turnip for feed, feed bit, turnip root, Silage, which is filled with lactic acid and fermented by lactic acid, hay, dried hay, herbage straw, straw of leguminous crops, leaves of leguminous plants, and the like. Special diets include mineral feeds such as fodder, rock salt, urea and its derivatives such as diuretic isobutane, nutrients such as diuretic isobutane, and nutrients that can be supplemented when only natural feed ingredients are mixed or supplemented with diets Feed additives, dietary supplements, which are substances added in small quantities, but are not limited thereto.

The composition for the feed comprising the composition for preventing or treating the insulin resistance-related disease according to the present invention can be prepared by adding the extract of Hymenoptera or its fraction at an appropriate effective concentration range according to various feed production methods known in the art.

The composition for feed according to the present invention is not particularly limited as long as it is an object for the purpose of prevention or treatment of an insulin resistance disease, and any composition can be applied. For example, it can be applied to any non-human animal such as a monkey, a dog, a cat, a rabbit, a guinea pig, a rat, a mouse, a cattle, a sheep, a pig,

Another aspect of the present invention provides a food composition for preventing or ameliorating an insulin resistance-related disease comprising an extract of Ornithoptera, or a fraction thereof, as an active ingredient.

The above extracts or fractions thereof, insulin resistance-related diseases are as described above.

Namely, the extract of Huaxiavin or its fractions can be added to food compositions for the purpose of preventing or improving insulin resistance-related diseases such as obesity, hyperlipemia, hypertension, arteriosclerosis, hyperinsulinemia, diabetes, liver disease and the like.

The food composition may comprise a pharmaceutically acceptable carrier.

The food composition of the present invention includes all forms of functional food, nutritional supplement, health food, and food additives, and the food composition of this type is known in the art And can be prepared in various forms according to known conventional methods.

When the extract of Huangshan Vine or a fraction thereof is used as a food additive, the Huangshanvine extract or its fraction may be used as it is, or may be used together with other food or food ingredients, and suitably 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). In general, when preparing a food or beverage, the extract is added in an amount of 0.0001 to 1% by weight, more preferably 0.001 to 0.1% by weight, of the raw material composition. However, in the case of long-term ingestion intended for health and hygiene purposes or for the purpose of controlling health, the amount can also be used in the above-mentioned range.

There is no particular limitation on the kind of the food. Examples of the food to which the above substances can be added include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include healthy foods in a conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Such natural carbohydrates are 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. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, and more specifically about 0.02 to 0.03 g per 100 of the composition of the present invention.

In addition to the above, the composition of the present invention may further contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acids and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, A carbonating agent used in a carbonated beverage, and the like. The proportion of such additives is not critical, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention. In addition, the composition of the present invention may contain flesh for the production of natural fruit juice, fruit juice beverage and vegetable beverage. The proportion of such flesh is not critical but is generally selected in the range of 0.01 to 10 parts by weight per 100 parts by weight of the composition of the present invention. These components can be used independently or in combination.

The present invention relates to a method for inhibiting the production of ROS caused by oxidative stress, inhibiting glutamyl oxaloacetic transaminase (GOT) and glutamyl pyruvate transaminase (GPT), which inhibit the absorption of triglycerides by inhibiting the absorption of triglycerides, Of the present invention is effective in the prevention and treatment of insulin resistance such as hyperinsulinemia, obesity, diabetes mellitus, hyperlipemia, arteriosclerosis, hypertension and liver disease, thereby preventing the prevention of insulin resistance-related diseases including hornblende extract or its fractions Or a composition for treatment. It can also be applied to food compositions for preventing or ameliorating insulin resistance-related diseases, livestock feeds or feed additives.

FIG. 1 is a graph showing a decrease in blood insulin concentration after administering a high-insulin-induced mouse to a hyperinsulinemia-induced mouse.
FIG. 2 is a graph showing that weight gain was suppressed by administering the extract of Hymenoptera to obesity-induced mice by the high fat diet method.
FIG. 3 is a graph showing that the impaired glucose tolerance was improved by administering the extract of Hansampigin to a db / db mouse having an impaired glucose tolerance.
Figure 4 is a graph showing db / db mice on a graph showing that the administration by the Humulus japonicus extract improves the insulin resistance.
FIG. 5 is a graph showing blood glucose lowering efficacy after administering Streptozotocin to a mouse induced diabetic rats with a single dose of Hansampigin extract.
FIG. 6 is a graph showing that the absorption of neutral fat in blood after the administration of the extract of Hornblende japonica to a mouse induced acute hyperlipemia by administration of corn oil.
FIG. 7 is a graph showing that the formation of atherosclerotic plaques and atherosclerotic plaques in the inner wall of the aorta was reduced by administering the extract of Hanssemide japonica to a mouse induced by arteriosclerosis using a mouse lacking apolipoprotein E;
FIG. 8 is a graph showing that the blood pressure is lowered after administering the extract of Hwangsan japonica to a rat having hypertension.
FIG. 9 is an image obtained by treating the liver cancer cell line induced by oxidative stress with tert -butyl hydroperoxide ( t- BHP) by treatment with Hanssemann japonica extract in a concentration-dependent manner and decreasing ROS.
FIG. 10 is a graph showing that GOT and GPT values, which are liver damage markers, in the plasma of animals administered with the extract of Panax Notoginseng to the mouse model in which hepatotoxicity was induced by carbon tetrachloride were significantly reduced.
FIG. 11 is a graph showing an immunofluorescence staining image showing a decrease in 4-HNE production, which is an index of lipid oxidation in animal liver tissues administered with Horseradish Vine Extract in a mouse model in which acute liver injury was induced by carbon tetrachloride, and a significantly reduced liver lipid peroxide concentration to be.
FIG. 12 is a graph showing a decrease in hepatic triglyceride by administering an extract of Hwangsan Jusung to a liver-induced mouse administered with a high fat diet.
FIG. 13 is a histopathological analysis showing that hepatitis was reduced by administering the extract of Hwangsan japonica to a mouse induced by hepatitis accompanied by fatty liver by ingesting a methionine-choline deficient feed.
FIG. 14 is a histopathological analysis chart showing that hepatocyte hepatocyte necrosis is remarkably reduced by administration of Conjugated Vine Extract in mice induced to concanavalin with interlobular hepatitis and hepatocyte necrosis.
FIG. 15 is a graph showing that alpha-smooth muscle actin (alpha -SMA), which is an index of liver fibrosis, is decreased by administering the extract of Hwangsan japonica in a mouse induced by cirrhosis by repeated administration of carbon tetrachloride.

Hereinafter, the constitution and effects of the present invention will be described in more detail through examples. These embodiments are only for illustrating the present invention, and the scope of the present invention is not limited by these embodiments.

Example  1. Preparation of Hwangsam Vine Extract

The resulting extract was filtered and concentrated, and the filtrate was concentrated in a deep freezer. The filtrate was concentrated, and the filtrate was concentrated. , The sample was frozen and dried using a freeze dryer. The extracted extracts of Hamsangbin were dissolved in 0.5% CMC (carboxy methylcellulose) solution and used in DMSO for hepatocyte experiment.

Example  2. Animal model of horseradish extract Hyperinsulinemia  Analysis of inhibition effect

8 week old male C57BL / 6 mice were maintained in the SPF breeding room controlled at the temperature of 22 ± 2 ° C, humidity of 55 ± 10% and 12 hours of illumination, and maintained for 8 weeks (Dyets, # 100244, USA ) Were administered orally once daily at a concentration of 125 mg / kg, 250 mg / kg, and 500 mg / kg, respectively. At the end of the experiment, blood was collected and plasma was separated and the insulin concentration was measured. As a result of the analysis, 2.57 ± 0.73 ng / ml in the 125 mg / kg group and 1.66 ± 0.26 ng / ml and 500 mg / kg in the 250 mg / kg group were administered compared with the control group of 7.3 ± 1.51 ng / The experimental group showed a blood insulin concentration of 1.61 ± 0.13 ng / ml, and all of the experimental groups treated with the Hansampigin extract showed a significant decrease in the insulin concentration, thus confirming the effect of the Hanssemark extract on the hyperinsulinemia inhibition One).

Example  3. Analysis of obesity inhibitory effect of hornblende extract on animal model

As described above, when 8-week-old male C57BL / 6 mice were fed with high fat diet (Dyets, # 100244, USA) for 8 weeks, diabetic obesity was induced in addition to hyperinsulinemia, The effect of oral administration at a concentration of 125 mg / kg, 250 mg / kg and 500 mg / kg was confirmed once in each case. As a result of comparing the body weights of the control and experimental mice, it was observed that weight gain was significantly inhibited during the entire experimental period from one week to 8th week in all the experimental groups treated with Hwangsan japonicum extract, Kg body weight of the control group was 28.3 ± 2.4%, 26.1 ± 2.7% of the 250 mg / kg group and 26.0 ± 2.4% of the 500 mg / kg group, respectively, 1.1%, indicating that the extract of Panax ginseng extract significantly reduced the weight gain induced by the high fat diet, thereby suppressing obesity (FIG. 2).

Example  4. Animal model of horseradish extract Glucose tolerance  Analysis of improvement effect

In general, db / db mice are animals with impaired glucose tolerance as a representative type 2 diabetes model animal. Six-week-old male db / db mice were allowed to consume 500 mg / kg of Horseshoe vinegar extract for 1 day for 1 week. The glucose tolerance test was used to evaluate the effect of the extract on the glucose tolerance test. Specifically, animals in the experimental group to which the control and hornblende extracts were administered were fasted for 16 hours, and glucose administered at a concentration of 1 g / kg was intraperitoneally injected. At 0, 15, 30, 60, 90 and 120 minutes Were observed.

At 30 minutes after glucose administration, the highest blood glucose level was observed in both groups. Blood glucose levels at 15 minutes and 30 minutes were higher than those of control group (572 ± 26 mg / dl, 726 ± 64 mg / dl) (463 ± 24 mg / dl, 529 ± 28 mg / dl), respectively. From these results, it was confirmed that the administration of the extract of Huangshan japonese improves the glucose tolerance by inhibiting the increase of blood glucose by injecting glucose (FIG. 3).

Example  5. Analysis of Insulin Resistance Improvement Effect of Horseradish Vine Extract Using Animal Model

Db / db mouse, which is a type 2 diabetes model of Example 4, exhibits typical insulin resistance in addition to impaired glucose tolerance. To confirm the efficacy of the insect resistance against insulin resistance, kg / kg diet for 1 week, then fasted for 4 hours for insulin tolerance test, and injected with 1 U / kg of insulin at 0, 30, 60, 90, and 120 minutes after the administration.

As a result of the analysis, there was no difference between the control group and the experimental group in the blood glucose level before insulin administration, but the blood glucose level decreased from the control group (295 ± 37 mg / dl) to the control group (308 ± 37 mg / dl) And this phenomenon lasted up to 120 minutes. From these results, it was confirmed that the administration of the hornblende extract increased the sensitivity to insulin, thereby improving insulin resistance (FIG. 4).

Example  6. Streptozotocin  Analysis of the effects of Horseradish Vine Extract on the diabetic animal model used

6-1. C57BL / 6 for mouse Streptozotocin ( streptozotocin ) Oral administration of intraperitoneal administration and extracts of hornblende

Streptozotocin (40 mg / kg) was administered intraperitoneally for 5 consecutive days to induce diabetes in male C57BL / 6 mice at 8 weeks of age. Two days after the average blood glucose level exceeded 200 mg / dl, the hornblende extract suspended in 0.5% CMC was orally administered at a concentration of 500 mg / kg once. During the experiment, feed intake and water were freely available to all animals. The animals were kept in an SPF environment controlled at a temperature of 22 ± 2 ° C, a humidity of 55 ± 10% and a light intensity of 12 hours.

6-2. Determination of blood sugar change effect in diabetic animal model

The mice that induced diabetes mellitus with streptozotocin in the above 6-1 were firstly collected with 0.5% CMC in the control group and once at a concentration of 500 mg / kg in the experimental group. Orally. Blood samples were taken from the mouse tail for 0.5, 1, 3, 6, and 12 hours after oral administration and blood glucose changes were observed.

As a result, there was no difference in the initial blood glucose level of the control group of 229 ± 11 mg / dl and the initial blood glucose level of the experimental group of 238 ± 13 mg / dl before the administration of the hanam hull extract. However, And 290 ± 13 mg / dl, respectively. The blood glucose level was decreased to 236 ± 12 mg / dl in the experimental group. From these results, it was confirmed that the administration of Horseradish Vine Extract effectively inhibited the pancreatic damage of C57BL / 6 mouse caused by streptozotocin and thus had a hypoglycemic effect by protecting the pancreas (FIG. 5).

Example  7. Analysis of administration effect of horseradish peroxidase extract in corn oil-induced acute hyperlipemia animal model

7-1. C57BL / 6 Oral administration of corn oil and hymenoptera extract to mice

8-week-old male C57BL / 6 mice were fasted for 16 hours, and then primary blood was collected and orally administered at a concentration of 100, 200 or 400 mg / kg once orally at a concentration of 100, 200 or 400 mg / kg suspended in 0.5% CMC. After 30 minutes of administration of the extract of Panax Notoginseng (100, 200 or 400 mg / kg) and the positive control of the fat inhibitor, Xenical (50 mg / kg), corn oil was administered at a concentration of 5 g per kg body weight ≪ / RTI > During the experiment, feed intake was limited and water was freely available to all animals. The animals were kept in SPF (specific pathogen-free) environment at temperature of 22 ± 2 ℃, humidity of 55 ± 10% and light intensity of 12 hours cycle.

7-2. Administration of Hwangsan Vine Extract C57BL / 6 Changes in blood triglyceride content in mice

Primary blood was drawn from C57BL / 6 mice fasted for 16 hours, and blood samples were collected after one or two hours of corn oil administration. The obtained blood was centrifuged at 10,000 rpm for 5 minutes and the plasma was separated and the absorbance was measured at 550 nm using a kit for analyzing triglyceride (Asan Pharm Co, Korea), and the absorbance of the neutral The concentration of triglyceride in the blood of the group treated with hornbush extract was calculated based on the concentration of fat.

As a result, as shown in Table 1, the mice were orally administered with corn oil and the serum triglyceride concentration after 1 hour of the administration of corn oil was 227 ± 14 mg / dl in the experimental group, Compared with the normal control group (140 ± 02 mg / dl). On the other hand, the concentration of triglyceride in the blood was inhibited by 151 ± 12 mg / dl in the positive control group, and the concentration of triglyceride in blood was 181 ± 20 mg / dl and 170 15 mg / dl and 157 +/- 09 mg / dl, respectively. After 2 hours from the administration of corn oil, the blood triglyceride concentration of the control group was 232 ± 17 mg / dl, while that of the control group was 163 ± 8.51 mg / dl in the Xenical treatment group and 205 ± 14 mg / dl in the control group, 161 ± 22 mg / dl and 156 ± 04 mg / dl, respectively. Based on the above results, the same or better neutral fat suppressing effect than the positive control group, Xenical, was obtained. From these results, it was found that the administration of the extract of Horsham Vinegar effectively inhibits acute hyperlipemia of C57BL / 6 mice induced by administration of corn oil (Fig. 6).

Example  8. Apolipoprotein  E ( ApoE Analysis of the effect of Horseradish Vine Extract on animal models of atherosclerosis using deficient mice

8 week old male ApoE deficient mice were fed a 1.25% cholesterol atherosclerotic diet (Dyets # 102571, USA) for 6 weeks. The mice were divided into two groups to receive atherosclerosis, 0.5% CMC solution in the control group, and 250 mg / kg of Oral vinegar extract suspended in 0.5% CMC in the experimental group. During the experiment, feed intake and water were freely available for all animals. The feeding conditions of the animal room were maintained in an SPF environment adjusted to the temperature of 22 ± 2 ° C, humidity of 55 ± 10% .

After 6 weeks, the experiment was completed and autopsy was performed. The arterial area from the origin of the aortic arch to the distal end of the abdominal aorta was separated and fixed with 10% neutral formalin for 24 hours or more. Atherosclerotic plaque) was stained with oil-red O stain, which was stained with red color, and analyzed by visual and image analysis program. As a result of analysis, it was confirmed visually that red ginseng was reduced in the inner wall of the artery in the group treated with 250 mg / kg of Hansampigin extract. Using an image analysis program, the oil-red O showed a 10.4 ± 0.01% increase in the area of red stained area compared to the control group (18.8 ± 0.04%), and the fat deposition of the inner wall of the artery (atherosclerosis Half) was significantly inhibited. From these results, it was confirmed that the administration of the hornblende extract effectively inhibited arteriosclerosis in the atherosclerotic induction model using ApoE-deficient mice (Fig. 7).

Example  9. Analysis of administration effects of Horseradish Vine Extract using hypertension animal model

To investigate the effect of Hwangsan extract on hypertension, 12-week-old male SHR rats (Spontaneously hypertensive rats), widely used as an animal model of hypertension, were treated with 500 mg / kg The blood pressures before and after administration of Hwangsam Vine extract were compared and analyzed. As a result of the analysis, it was confirmed that the hornblende extract had an activity of inhibiting hypertension when the hornblende extract was lowered to 167 ± 8 mmHg one week after the administration of the hornblende, compared with the systolic blood pressure of 191 ± 6 mmHg before the administration of the hornbill.

Example  10. Liver cancer cell line  Active oxygen of extract Inhibition  analysis

Oxidative stress was induced by treatment with 300 μM t -BHP ( tert- butylhydroperoxide) in HepG2 cells of hepatocyte-host HepG2 cells for 3 h at concentrations of 0, 200, 400 and 800 ㎍ / ml. One hour later, to measure reactive oxygen species (ROS) occurring in the cells, the medium was replaced with a medium containing 10 μM DCF-DA (2 ', 7'-Dichlorofluorescein diacetate) and further incubated for 60 minutes ROS produced by fluorescence microscopy was analyzed.

As a result, it was observed that the degree of ROS production in hepatocyte by t- BHP was decreased as the concentration of horseradish peroxidase extract increased. From these results, it was confirmed that the hornblende extract had a protective action against oxidative stress (FIG. 9).

Example  11. Acute Liver damage  Animal model of horseradish vine extract Liver protection  Effect and lipid peroxide generation Inhibition  analysis

11-1. C57BL / 6 mouse Hepatotoxicity  Oral administration of Tricholoma matsutake extract

To investigate the liver protective effect of the extracts of Hamsanchus javanica in an animal model of acute liver damage, male C57BL / 6 mice of 8 weeks old were orally administered once at a concentration of 500 mg / kg in 0.5% CMC. Respectively. Silymarin was administered at a dose of 300 mg / kg as a positive control. After 30 minutes of administration, the mice were intraperitoneally injected with 1 ml / kg of carbon tetrachloride and corn oil (CCl ₄ : corn oil = 1: 1 v / v) and fasted for 24 hours. Blood and liver tissues were collected Respectively. During the experiment, water was freely available to all animals. The animals were kept in an SPF environment controlled at a temperature of 22 ± 2 ° C, a humidity of 55 ± 10% and a light intensity of 12 hours.

11-2. Liver damage  In the animal model, the blood of the hornblende extract GOT  And GPT  Effect analysis

In order to investigate the effect of Hansampigin extract on glutamyl oxaloacetic transaminase (GOT) and glutamyl pyruvic transaminase (GPT), which are representative indicators of liver damage in blood, in liver model mice induced by carbon tetrachloride as described above, Blood was collected from a retro-orbital sinus using a heparin-treated capillary tube and then centrifuged at 3,000 rpm for 10 minutes to obtain plasma from the upper layer. An automatic blood analyzer (Hitachi 7150, Japan).

As a result, the concentrations of GOT and GPT in the blood were 10292 ± 722 IU / L and 20905 ± 2455 IU / L in the experimental group, whereas 6824 ± 2268 IU / L and 12288 ± 2506 IU / , Which is significantly lower than that of the experimental control group, which is similar to that of the silymarin group used for the treatment of liver diseases. From these results, it was confirmed that the administration of the extract of Hwangsam was not only effective in inhibiting hepatocyte injury caused by carbon tetrachloride, but also had a protective action against the liver (FIG. 10).

11-3. Liver damage  Animal model of horseradish vine extract Liver tissue  of mine Lipid oxidation  The effect of 4-HNE (4-Hydroxynonenal) on the indicator

4-HNE, a commonly used lipid oxidation index, was stained with immunofluorescent staining to confirm the degree of lipid oxidation in liver tissue produced by carbon tetrachloride. After the administration of Horseradish Vine Extract and Carbon Tetrachloride, liver tissues were cut from each animal, fixed with 10% neutral formalin for 24 hours, dehydrated with ethanol, embedded in paraffin, Thick tissue slices were prepared and stained slides were made. 4-HNE antibody, an indicator of lipid oxidation, was used as a primary antibody for immunofluorescence staining. The secondary antibody was stained with Alexa Fluor 488 nm goat anti-rat IgG (1: 200) and observed with a fluorescence microscope. As a result, lipid oxidation was generally observed in the liver of the experimental group administered with carbon tetrachloride alone, but the lipid oxidation was remarkably improved in the experimental group treated with silymarin and hanam extract (FIG. 11A). Furthermore, the hornblende had lower lipid peroxides than silymarin, the control group. These results support the efficacy of Hwangsan japonicus extract in the treatment of liver disease.

11-4. Liver tissue  Quantitative analysis of lipid peroxides

In order to substantially measure the lipid peroxide content in liver tissue produced by carbon tetrachloride, 50-100 mg of liver tissue was suspended in PBS, homogenized with a homogenizer, centrifuged at 10,000 g for 5 minutes to obtain supernatant, 8% SDS was added to the supernatant and mixed well. A 1: 1 solution of 0.8% TBA and acetic acid (pH 3.5) was added and the mixture was heated at 100 ° C for 1 hour. After heating, centrifugation was carried out at 1,600 g for 10 minutes and absorbance was measured at 532 nm. As a standard solution, the content of lipid peroxide produced using 1,1,3,3-tetramethoxypropane was calculated and finally divided by protein content and expressed as μM / mg protein. The lipid peroxidation in liver tissues was 5.8 ± 2.1 μM / mg protein in the control group, 26.7 ± 6.4 μM / mg protein in the control group and 7.9 ± 3.3 μM in the silymarin treated group / mg protein, indicating that the production of lipid peroxides was significantly decreased in the group treated with the extract of Hymenoptera (Fig. 11B).

Example  12. Analysis of administration effect of horseradish peroxidase extract in fatty liver model

High density lipoprotein (125 mg / kg) was obtained from an 8-week-old male C57BL / 6 mouse for 8 weeks in a high fat diet (Dyets, # 100244, USA) 250 mg / kg and 500 mg / kg once a day. At the end of the experiment, the liver was sampled and pulverized with physiological saline. The fat component was extracted with a solution containing chloroform and methanol in a ratio of 1: 1, centrifuged, , Korea) was used to measure the triglyceride concentration.

The results showed that the concentration of triglyceride in the liver of the control group was 8.4 ± 0.7 mg / g Liver and 8.9 ± 0.7 mg / g liver, respectively, compared with 11.8 ± 1.6 mg / g Liver The concentration of hepatic triglyceride in liver was significantly decreased in all experimental groups treated with 0.8 mg / g liver and 500 mg / kg liver, and 8.3 ± 0.4 mg / g Liver. It was confirmed that there was an effect of suppressing fatty liver (Fig. 12).

Example  13. Methionine-choline deficiency Diet  Analysis of administration effect of Hwangsan japonicus extract in hepatitis hepatitis animal model

In order to investigate the effects of the extract of Hwasan japonica in hepatitis animal models with fatty liver using methionine - choline deficient diets, male C57BL / 6 mice at 8 weeks of age were treated with 0.5% CMC and a methionine - Experiments were carried out for 4 weeks by dividing the test group of Ornithoptera japonica extract suspended in 0.5% CMC at a concentration of 500 mg / kg. After the end of the experiment, the blood was collected and liver tissue was obtained and fixed with 10% neutral formalin for 24 hours or longer in order to compare the degree of hepatitis induced by methionine-choline deficient diet. Cyanine-eosin staining was performed and then observed with an optical microscope. A comparative analysis of hepatitis production was performed in accordance with the method reported in the previous literature (Kleiner et al., Hepatology 41: 1313-1321 (2005)), in which the inflammatory cells gathered in the microscopic field, foci) (the arrows in Fig. 13), and when there are no observations at all, 0, 1 or more is less than 2, 1 is less than 2, 4 is less than 2, And the scores obtained by observing the liver tissues of the control group and the group treated with the extract of Hwangsan japonicus were analyzed.

As a result, the hepatitis score obtained in the control group was 1.5 ± 0.08, whereas in the group treated with the extract of Hansampigin, 0.5 ± 0.10 was obtained, indicating that the extract of the Hanssung japonica effectively inhibited hepatitis accompanied by fatty liver induced by the methionine-choline deficient diet (Fig. 13A). At the same time, the concentrations of GOT and GPT, which are indicators of liver damage in the blood, were 289 ± 42 IU / L and 474 ± 60 IU / L, respectively, in the control group, 19 IU / L and 143 ± 23 IU / L, respectively, which were significantly lower than those of the control group.

From these results, it was found that the administration of the extract of hanamnia japonensis inhibited the infiltration of inflammatory cells in the liver due to the methionine-choline deficient diet, thereby preventing hepatitis and protecting the liver (Fig. 13B).

Example  14. Konkanavalin  A ( Concanavalin  A) induced Nonfat  Analysis of administration effect of Horseradish Vine extract on hepatitis animal model

To investigate the effect of Conjunctiva A (ConA) on hepatitis without hepatic failure, male C57BL / 6 mice at 8 weeks of age were treated with 0.5% CMC and 0.5% CMC Was administered once at a concentration of 500 mg / kg of Conjugate extract. After 30 minutes, ConA, which was diluted to 15 mg / kg with PBS, was administered intravenously. After 6 hours, blood and liver tissues were collected. In order to compare the degree of hepatocyte necrosis induced by ConA in hepatocytes, the cells were fixed with 10% neutral formalin for 24 hours or more, dehydrated with ethanol, embedded in paraffin, A tissue slice with a thickness of ㎛ was prepared and a slide for dyeing was made, and hematocylin and eosin staining were performed and observed with an optical microscope.

As a result of analysis, it was found that a wide range of acute hepatitis necrosis was observed throughout the liver tissues in the liver of the control group administered with ConA alone, while the hepatocyte necrosis was remarkably improved in the experimental group treated with the hornblende extract (Fig. 14 Dotted line). When the necrotic area was compared with the image obtained by using the image program, when the degree of damage of the control group was regarded as 100%, it was confirmed that it was significantly decreased in the group treated with the hornblende extract (FIG. 14A).

The concentrations of GOT and GPT in the blood of the control group and the group treated with the extract of Panax notoginseng extract were 5595 ± 397 IU / L and 7891 ± 834 IU / L in the control group, respectively. And 2880 ± 507 IU / L and 3428 ± 834 IU / L, respectively, in one group.

From these results, it was found that administration of the extract of Panax notoginsa effectively inhibited hepatitis caused by ConA, thereby effectively reducing liver damage (Fig. 14B).

Example  15. Analysis of administration effect of Horseradish Vine Extract on Cirrhosis-induced animal model using carbon tetrachloride

In order to investigate the effect of Hansampigin extract on liver cirrhosis, 8 week old male C57BL / 6 mice were treated with corn oil and carbon tetrachloride twice a week and treated with 0.5% CMC, Were administered to a 0.5% CMC suspension containing 500 mg / kg of Hansampigin extract for 4 weeks. For the induction of chronic cirrhosis, carbon tetrachloride was added to corn oil at a rate of 1 ml / kg for 1 week, 1.5 ml / kg for 2 weeks, and 2 ml / kg for 3 and 4 weeks. Respectively. After the end of the experiment, RNA was extracted from liver tissue to synthesize alpha-smooth muscle actin (α-SMA) gene, which is well known as liver cirrhosis index. CDNA was synthesized and real-time PCR analysis was performed.

As a result of analysis, it was confirmed that when the control group was 100%, the expression of the? -SMA gene was reduced to 64% of the control group by the horseradish peroxidase extract administration group (FIG. 15A).

In addition, western blot analysis was performed to analyze the effect of Hansampigin extract on α-SMA protein expression as well as gene expression. The liver tissues were taken from the control and the experimental groups, and were pulverized with a tissue lysis buffer, centrifuged at 13,000 rpm for 15 minutes, and the supernatant was obtained. Proteins were quantified by the Bradford method and the same amount of protein was dissolved in 10% And electrophoresed on acrylamide gel.

After electrophoresis, the cells were transferred to a nitrocellulose membrane using semi-dry transfer, blocked with 5% skim milk, and incubated with α-SMA (α-smooth muscle actin, abcam, UK) Overnight at 4 ° C. After washing, the cells were reacted with anti-goat IgG conjugated with peroxidase (Jackson ImmunoResearch, USA) for 1 hour at room temperature.

As a result of analysis, the expression of α-SMA protein was decreased to such an extent that the bands of the experimental group treated with Hwangsan japonicum extract could be distinguished visually, compared with the control group. As a result of quantitative analysis, when the control group was taken as 100% The vinegar extract administration group was reduced to 58% of the control group (Fig. 15B).

From the above results, it was confirmed that the extract of Huaxia japonica inhibits the expression of? -SMA, which is known to play the most important role in liver cirrhosis in an animal model of liver cirrhosis induced by carbon tetrachloride, thereby preventing cirrhosis.

Example  16. Statistical analysis

All values are expressed as mean ± standard error (SEM). Statistical significance was set at 0.05 for statistical significance, and the Tukey-Kramer HSD test was used for multiple comparisons. The comparisons between groups were made by Student t-test.

These results suggest that the extract of the present invention or fractions thereof is effective for the prevention and treatment of diseases of various categories of insulin resistance-related diseases.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

Claims (16)

A disease selected from the group consisting of atherosclerosis, nonalcoholic fatty liver disease, hepatic hepatitis, nonfat hepatitis, end-stage fibrosis liver disease, nonviral chronic hepatitis, cirrhosis, acute liver injury and liver cancer, ≪ / RTI >
The composition according to claim 1, wherein the extract is extracted with a solvent selected from the group consisting of water, an alcohol having 1 to 6 carbon atoms, and a mixed solvent thereof.
delete delete delete delete delete delete The pharmaceutical composition of claim 1, wherein the composition further comprises a pharmaceutically acceptable carrier.
The composition of any one of claims 1, 2, or 9 is administered to a mammal for the treatment of atherosclerosis, nonalcoholic fatty liver disease, hepatic liver disease, nonfat hepatitis, end-stage fibrosis liver disease, nonviral chronic hepatitis, cirrhosis, To a subject other than a human suspected of having a disease selected from the group consisting of < RTI ID = 0.0 >
Atherosclerosis, nonalcoholic fatty liver disease, hepatic hepatitis, nonfat hepatitis, end-stage fibrosis liver disease, non-viral chronic hepatitis, liver cirrhosis, acute liver injury and liver cancer.
A disease selected from the group consisting of atherosclerosis, nonalcoholic fatty liver disease, hepatic hepatitis, nonfat hepatitis, end-stage fibrosis liver disease, nonviral chronic hepatitis, cirrhosis, acute liver injury and liver cancer, A quasi-composition for prevention or improvement.
A disease selected from the group consisting of atherosclerosis, nonalcoholic fatty liver disease, hepatic hepatitis, nonfat hepatitis, end-stage fibrosis liver disease, nonviral chronic hepatitis, cirrhosis, acute liver injury and liver cancer, A composition for feed additives or feeds for prevention or improvement.
A disease selected from the group consisting of atherosclerosis, nonalcoholic fatty liver disease, hepatic hepatitis, nonfat hepatitis, end-stage fibrosis liver disease, nonviral chronic hepatitis, cirrhosis, acute liver injury and liver cancer, ≪ / RTI >
14. The composition of claim 13, wherein the food composition further comprises a pharmaceutically acceptable carrier.
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