KR20120060192A - Composition for ameliorating the ER stress or the mitochondrial dysfunction comprising the extract of Angelica dahurica or imperatorin - Google Patents

Abstract

PURPOSE: A pharmaceutical composition or food composition containing Angelica dahurica extract or imperatorin is provided to prevent or treat diseases caused by ER stress or mitochondrial dysfunction. CONSTITUTION: A pharmaceutical composition for preventing or treating obesity, body fat increase, steatosis, liver fibrosis, liver disease, pancreas, hypertension, artery scleroma, cardiovascular disease, or cancer contains Angelica dahurica extract and pharmaceutically acceptable carrier. The pharmaceutical composition contains imperatorin or pharmaceutically acceptable salt thereof and pharmaceutically acceptable carrier. A medicinal formulation containing the pharmaceutical composition is manufactured in a form of tablets, oral tablets, liquids, jelly, aqueous or oil suspension, granules, emulsions, soft or hard capsules, or syrup or elixir.

Description

Composition for ameliorating the ER stress or the mitochondrial dysfunction comprising the extract of Angelica dahurica or imperatorin}

The present invention is white paper (白芷, Angelica dahurica ) extract or imperatorin (imperatorin) as an active ingredient in the endoplasmic reticulum stress (ER stress) or mitochondrial dysfunction improvement useful pharmaceutical composition or food composition.

In the endoplasmic reticulum, there are rough ER (RER) and smooth ER (SER) to which ribosomes are attached. The main function of the rough endoplasmic reticulum is protein synthesis, and about one-third of the intracellular proteins are synthesized by the rough endoplasmic reticulum. However, the smooth endoplasmic reticulum is found in most cells, and plays an important role in synthesizing various lipids and steroid hormones and regulating the intracellular calcium concentration as a calcium reservoir. When calcium homeostasis, glycosylation or disulfide bond formation in the endoplasmic reticulum is not properly formed due to various physiological or pathological conditions, protein folding does not occur properly. Unfolded proteins that exceed the ability of the endoplasmic reticulum to process are accumulated or calcium is depleted, resulting in functional impairment of the endoplasmic reticulum. This condition is called endoplasmic reticulum stress (Zhao & Ackerman, 2006; Mikyung Kim and Geunkyu Park, 2008). The endoplasmic reticulum stress (ER stress) increases apoptosis and the formation of a large amount of reactive oxygen species (ROS) through several signaling, leading to damage to cells.

Diseases caused by endoplasmic reticulum stress include neurodegenerative disease, bipolar disorder, diabetes mellitus, atherosclerosis, inflammation, ischemia, and heart disease. diseases), liver diseases, pancreatic diseases, cancer, etc., and are known to be involved in the occurrence of various diseases, and in particular, the association with obesity and diabetes has begun to be reported recently. These findings suggest that relieving endoplasmic reticulum stress could prevent/treat the occurrence of metabolic syndrome including diabetes (Banhegyi et al., 2007; Lindholm et al., 2006; Tsiotra & Tsigos, 2006; Yoshida, 2007).

In general, mitochondria are organelles that exist within cells and are known as "cells' powerhouses." Mitochondria not only produce ATP in cells, but also perform essential functions for maintaining the life of cells/individuals such as apoptosis, ion homeostasis, sugar and fat metabolism, and pyrimidine/urea/heme synthesis. do.

Mitochondrial abnormalities are also correlated with aging-related degenerative diseases-cardiovascular disease, Parkinson's disease, Huntington's disease, dementia and spinal cord injury. In other words, in these disease models, the mitochondrial electron micrographs are swelling and enlarged compared to normal, or structural modifications such as loss of the cristae structure, and the number of mitochondrial DNA (mtDNA) per cell are also reduced. A case in which mtDNA was mutated was also observed. When monitoring mitochondrial function changes according to aging in humans and animals, the number of copies of mitochondrial DNA (mtDNA) and mtRNA decreased as aging progressed (Barazzoni et al., 2000), and Attardi et al. It has been reported that T414G and T408A mutations accumulate in the D-loop, which is a regulatory site for mtDNA replication and transcription (Michikawa et al., 1999; Wang et al., 2001). Recently, it has been reported that substances identified as factors causing the onset of such neurodegenerative diseases exist in mitochondria or are closely related to mitochondrial function impairment (Beal, 1995; Chen & Yan, 2007; Scheffler, 2008; Wallace, 2005).

In the past, genetic studies on mitochondria related to human diseases have proven that mutations in mitochondrial genes cause metabolic and neurological related genetic diseases (Wallace, 2005). Recent research results reported by various laboratories, including the inventors' laboratories of the present invention, show that mitochondrial function impairment due to genetic and environmental causes is metabolic syndrome (obesity, diabetes, lipid metabolism, hypertension). It has been shown that the above symptoms are a disease that occurs simultaneously in one person and are directly related to chronic inflammatory diseases including insulin resistance (insulin resistance), neurodegenerative diseases, and the aging process. For example, insulin resistance in elderly people is correlated with lean body mass and fat mass, and mitochondrial oxidative phosphorylation activity in the elderly as measured by in vivo NMR technique. As this is about 40% lowered, it was confirmed that the decrease in mitochondrial function is a causative factor of insulin resistance (Pettersen et al., 2003). In addition, it has been reported that PGC-1α, a co-activator that enhances the activity of the ligand of peroxisome proliferators activated receptor γ (PPARγ), which is used as a new therapeutic agent for insulin resistance, promotes mitochondrial production. Therefore, it is accepted that a definite causal relationship is established between mitochondrial function and the onset of insulin resistance and metabolic syndrome.

Overweight and obesity are a state in which the proportion of fat tissues among the components of the body has increased beyond normal, and is not only a risk factor for various diseases, but is also evaluated as a health risk factor. Active management is required. Although it is a single disease, it is a direct or indirect cause of various and serious health problems. In the busy life of modern people, the intake of foods containing high calories and a large amount of fat and salt is increasing due to the improvement of dietary life and westernization, while the intake of foods containing a large amount of fat and salt is increasing, while the amount of activity is rather decreased, leading to an increase in the frequency of obesity (Kim et al., 2007; Lee Yun-na. Et al., 2004). As a result, the cost of socioeconomic losses is increasing rapidly (Wolf & Colditz, 1998). As a result of a domestic epidemiological survey, it is reported that the prevalence of obesity is more than twice as high in the underprivileged of low-income and low-educated people like the West, and various chronic diseases are increasing due to this. According to recent studies, it is reported that overweight and obesity have a high burden of diseases such as diabetes, ischemic heart disease, and stroke. In particular, Asians have a lower standard of obesity than foreigners, and in Korea, the problem of overweight is more serious than that of foreign obesity (Jeffery & French, 1997; Must et al., 1999; Kang Jae-Hun; And Namsoon Kim, 2002; Youngseol Kim, 1990; Hyemi Choi et al., 2004).

When both parents are obese in an environment where activity is low and high-calorie food is consumed, the parent's eating habits are transmitted to the child, and 70-80% of the children are obese. In addition, in the case of adult obesity, the size of adipocytes increases, but in the case of child obesity, the number and size of adipocytes increase, so child obesity is more dangerous than adult obesity, and various diseases including the incidence of adult diseases when obese children become adults. Since the risk of the disease increases, it is urgent to prevent or treat it (Champ et al., 2005). As such, overweight and obesity are serious problems that require active social management beyond individual problems, and recently, the World Health Organization (WHO) recognizes that obesity, which spreads like an infectious disease, is a big problem enough to define it as a disease (World Health Organization, 2004).

Diabetes is largely classified into type 1 and type 2 diabetes, and type 1 diabetes occurs in children or puberty, and is caused by deficiency of insulin due to a destructive lesion of β cells in the pancreas. In addition, type 2 diabetes develops after age 35 and is caused by decreased insulin secretion and insulin resistance (cell resistance to insulin) in which the liver, muscle, fat cells, and pancreas, which are insulin sensitive peripheral tissues, do not respond to insulin. In this disease, due to insufficient insulin or insulin resistance, the glucose tolerance to maintain blood sugar levels despite food intake decreases, so that high blood sugar is maintained for a long period of time, resulting in diabetes, in which sugar is excreted in the urine. Early symptoms of diabetes include polyuria, thirst, and decreased ability to maintain blood sugar, resulting in increased appetite and insulin resistance, leading to inadequate use of sugar, leading to weight loss (Champ et al., 2005; National Institutes of Health, 2004). .

Drugs that can prevent and treat various symptoms of metabolic syndrome, including obesity and diabetes, have not yet been reported, and in the case of drug therapy drugs, most drugs aimed at alleviating symptoms. Troglitazone, recently used as a treatment for insulin resistance In the case of the PPARγ ligand of the line, it is observed that the cure rate is low in Koreans, and its use is still limited due to side effects such as secondary obesity and edema, which may appear due to the property of promoting adipogenesis. Therefore, if a drug that can prevent/recover mitochondrial function damage and/or endoplasmic reticulum stress and/or (and/or) mitochondrial function damage, which is pointed to as a cause of diabetes/obesity/neurodegenerative diseases with low side effects, is developed, environmental or genetic factors It will be possible to develop a therapeutic agent that can prevent/treat metabolic syndrome and related degenerative diseases caused by. These drug candidates should be excellent in relieving endoplasmic reticulum stress and improving mitochondrial function, and should also have an effect of reducing appetite and unnecessary body fat and improving insulin resistance. However, no substance has yet been reported to have such an effect.

Meanwhile, a blank paper ( Angelica dahurica ) is a 2-3-year-old herb that grows naturally in Korea, China, and Japan. In oriental medicine, the herbal medicine made by drying this root is called “baekji”, and its main physiologically active substances are imperitoline and isoimperatorin. ), oxypeucedanin, phellopterin, and byakangelicol. In oriental medicine, many white papers have been known for their effectiveness. Especially, they are used to treat sweating, soothing, pain, cold, headache, and toothache, and are known to have whitening, pore contraction, and anti-inflammatory effects. However, there is no known efficacy related to endoplasmic reticulum stress or mitochondrial function, insulin resistance, obesity, diabetes and fatty liver.

Regarding a pharmaceutical composition containing white paper extract and imperatorin as an active ingredient, for example, Korean Patent Registration No. 10-0527876 says “Convulsive properties containing imperatorin and/or falcarindiol as active ingredients. Under the name of "a composition for the prevention and treatment of diseases", "a pharmaceutical composition for the purpose of anticonvulsant treatment and prevention with less side effects of imperatorin and/or falcarindaiol distributed and extracted from the roots of copper sticks" is proposed. As for other Korean patents, Registration No. 10-036152 is proposed as “a white paper, gobon extract, and a cosmetic composition containing this extract.” However, the composition shows the occurrence of fatty liver and metabolic syndrome through the improvement of mitochondrial function. There is no suggestion of the efficacy of suppressing or reducing the occurrence of metabolic syndrome and improving symptoms in obese patients, diabetic patients, and aging.

All the contents of the prior documents cited in the specification of the present invention are introduced into the present specification. In addition, the information described herein is only intended to help an understanding of the technical background of the present invention, it is natural that it cannot act as a prior art for the present invention.

Most of the treatments for diseases such as insulin resistance, obesity, diabetes and fatty liver alleviate the symptoms of the disease. Accordingly, an object of the present invention is to provide a pharmaceutical composition having an effect of alleviating or improving endoplasmic reticulum stress and mitochondrial function damage, which are pointed out as the cause of the disease.

In order to solve the above problems, the inventors of the present invention have conducted ceaseless studies, surprisingly, for the first time that natural materials such as white paper extract and imperatorin alleviate or improve endoplasmic reticulum stress and mitochondrial function damage, and provide them as a preventive or therapeutic agent for related diseases. do.

The composition containing the white paper extract or imperatorin of the present invention as an active ingredient has the effect of relieving endoplasmic reticulum stress and/or improving mitochondrial function. Therefore, the composition of the present invention can be used as a pharmaceutical composition or a food composition for relieving stress of the endoplasmic reticulum and/or improving mitochondrial function.

In addition, the present invention can also be used as a kit for providing a method of reducing stress of the endoplasmic reticulum and/or improving the function of mitochondria using a white paper extract or imperatorin.

Therefore, the pharmaceutical composition of the present invention is associated with relieving stress of the endoplasmic reticulum and/or dysfunction of mitochondria, obesity, insulin resistance, increased body fat, fatty liver, hepatic fibrosis, metabolic syndrome, lipid metabolism, hypertension, neurodegenerative diseases, bipolar disorder. It can be used for the prevention or treatment of disorders, diabetes, arteriosclerosis, ischemia, heart disease, liver disease, pancreatic disease, cancer, aging, cardiovascular disease, Parkinson's disease, Huntington's disease, dementia or spinal cord injury.

1 shows the effect of white paper extract (ADEx) or imperatorin (IMP) on apoptosis of Hep G2 cells by endoplasmic reticulum stress induced by thapsigagin or tunicamycin.
Figure 2 is a result showing the inhibitory effect of white paper extract (ADEx) on the expression of the endoplasmic reticulum stress marker genes GRP78 and XBP1p expressed by endoplasmic reticulum stress in Hep G2 cells.
3 is a result showing the inhibitory effect of imperatorin (IMP) on the expression of endoplasmic reticulum stress marker genes GRP78 and XBP1p in Hep G2 cells.
Figure 4 is a result showing the effect of imperatorin (IMP) on the reduction of the amount of ATP by endoplasmic reticulum stress (tapshigagin or tunicamycin) in Hep G2 cells.
5 is a result showing the effect on the amount of change in body weight when the white paper extract (ADEx) was administered to diabetic mice.
6 is a result showing the effect on the weight of white fat of the testis by administration of white paper extract (ADEx) to diabetic mice.
7 is a result of measuring and confirming the amount of oxygen consumption, which is mitochondrial activity, in the soleus muscle of mice to which white paper extract (ADEx) was administered with an Oxygraph-2k instrument.
8 is a result showing the effect of administration of white paper extract (ADEx) in the oral glucose tolerance test of Lean mice and diabetes model (db/db) mice.
9 is a result of measuring the change in dietary intake by administration of imperitoline in mice in the high-fat diet group and the low-fat diet group.
10 shows the effect of imperatorin on the expression of an appetite promoting gene in the hypothalamus (hypothalamus) of the brain of mice in the low-fat diet group and the high-fat diet group.
11 shows the effect of imperatorin on the weight of white fat in mice in the low-fat diet group and the high-fat diet group.
12 is a result of confirming the effect of imperatorin on C/EBP-α expression in the liver of mice in the low-fat diet group and the high-fat diet group by immunohistochemistry.
13 is a result of confirming the morphology of cells according to whether imperiitoline was administered or not by using H&E staining in the liver of mice in the low-fat diet group and the high-fat diet group.
14 is a result of measuring the amount of mitochondrial oxygen consumption in the soleus muscle collected from mice in the low-fat diet group and the high-fat diet group using the Oxygraph-2k instrument (Oroboros, Austria) according to the administration of imperatorin.
FIG. 15 is a result of measuring oral glucose tolerance according to whether imperitoline was administered in mice in the low-fat diet group and the high-fat diet group.

The present invention relates to a pharmaceutical composition or a food composition for relieving stress of the endoplasmic reticulum and/or improving mitochondrial function using white paper extract or imperatorin as an active ingredient.

The present invention also provides a method for relieving stress in the endoplasmic reticulum and/or improving the function of mitochondria using white paper extract or imperatorin, and a kit therefor.

The inventors of the present invention believe that inducing endoplasmic reticulum stress in Hep G2 cells causes apoptosis, but pretreatment with white paper extract reduces apoptosis (Fig. 1), and expression of endoplasmic reticulum stress marker genes is reduced by white paper extract or imperatorin. It was found to decrease (Figures 2 and 3). In addition, it was observed that inducing endoplasmic reticulum stress in Hep G2 cells decreases the amount of intracellular ATP, but significantly increases the amount of ATP when imperatorin is treated (Fig. 4).These results indicate that imperatorin relieves endoplasmic reticulum stress. This is thought to be due to increased mitochondrial activity and ATP production.

In addition, the inventors of the present invention administered white paper extract (ADEx) to diabetes model mice (db/db mice) and lean mice for 33 days and observed changes in body weight, although no change in dietary intake of each group was observed, In the case of lean mice, the weight gain was not significantly affected by the white paper extract, but in db/db mice, the weight gain decreased when the white paper extract was administered (Fig. 5). In addition, in the case of db/db mice, the amount of white fat in the testis was significantly reduced when the white paper extract was administered (Fig. 6). Administration of white paper extract increased mitochondrial activity of soleus muscle in mice in both lean and db/db mice. In particular, administration of the white paper extract increased the activity of the oxidative phosphorylation (OXPHOS) complex to a remarkably high level in the case of disease (db/db mice) than in the normal state (Fig. 7). In the oral glucose tolerance test, db/db mice showed about 4-5 times higher blood sugar than the control group, but when the white paper extract was administered, the insulin sensitivity was increased by the white paper extract, so that blood sugar decreased compared to the db/db group (Fig. 8).

The inventors of the present invention found that the weight and dietary intake of mice in the high-fat diet group increased compared to mice in the low-fat diet group, but when imperatorin (IMP) was administered, the weight gain and the increase in dietary intake were decreased (Tables 1 and 2, Fig. 9). This decrease in dietary intake is that the mRNA of NPY (neuropeptide Y) and AgRP (agouti-related protein), which are hypothalamic appetite-enhancing neuropeptides, was increased due to high-fat diet intake, and their expression was decreased due to imperatorin administration. As shown in (Fig. 10), it appears that imperatorin exhibits an appetite suppressing effect by inhibiting the expression of NPY and AgRP. In addition, the present inventors found that excessively accumulated fat along with weight gain due to intake of a high fat diet significantly decreased at high concentrations when imperatorin was administered together (Figure 11), imperatorin accumulated by a high fat diet. It has been shown to be effective in reducing body fat. In addition, fatty liver is formed due to excessive intake of fat.According to the present inventors' immunohistochemistry, excessive expression of C/EBP-α caused by ingestion of a high-fat diet is when imperatoline is administered. It was normalized to a level similar to that of the control group (FIG. 12), and when many lipid droplets of the group ingesting a high-fat diet were dose-dependently normalized to a form of hepatocytes similar to those of the control group (FIG. 13). This result shows that imperatorin inhibits the synthesis or accumulation of fat in the liver due to ingestion of a high fat diet and has excellent efficacy in restoring liver function. In addition, it was observed that the administration of imperitoline in the high-fat diet group improved the mitochondrial activity of the muscles, especially the activity of OXPHOS complex I (Figure 14), and it was observed that fasting blood sugar slightly decreased and the blood glucose clearance rate increased after glucose intake. Observed (Fig. 15).

therefore. The white paper extract or imperatorin of the present invention reduces the risk of obesity by 1) reducing increased body weight and appetite due to genetic or environmental factors. 2) By improving mitochondrial function through relieving endoplasmic reticulum stress, it strongly improves insulin resistance and helps glucose present in the blood to be absorbed into each tissue in a short time. 3) By reducing the weight of white adipose tissue, it suppresses unnecessary body fat increase, thereby inhibiting or reducing the accumulation of fat in each tissue. In addition, 4) has the effect of inhibiting or reducing fatty liver or liver fibrosis. Therefore, if the composition containing the white paper extract and imperatorin according to the present invention is used, it exhibits a strong effect to prevent various diseases that may be caused by irregular eating habits and insufficient exercise amount, and thus, metabolic syndrome and the accompanying fatty liver prevention and treatment As can be used as a pharmaceutical composition.

In addition, the white paper extract or imperatorin of the present invention can be used for the prevention, improvement or treatment of diseases caused by endoplasmic reticulum stress or mitochondrial function impairment. Diseases that can be prevented or treated include, for example, obesity, insulin resistance, increased body fat, fatty liver, liver fibrosis, metabolic syndrome, lipid metabolism, hypertension, neurodegenerative diseases, bipolar disorder, diabetes, arteriosclerosis, ischemia, heart disease. , Liver disease, pancreatic disease, cancer, aging, cardiovascular disease, Parkinson's disease, Huntington's disease, dementia or spinal cord injury, and the like, but is not limited thereto.

In the present invention, "treatment" means inhibition and alleviation of disease progression. Terms that are not otherwise defined in the present specification have the meanings commonly used in the technical field to which the present invention pertains.

In the present invention, the white paper extract and imperatorin may be obtained by distribution and extraction with an organic solvent (for example, n-butanol, methanol, ethanol, chlorform, etc. may be used, but is not limited thereto) and water from the white paper root. There is .

Imperatorin of the present invention can be used in free form or in the form of a pharmaceutically acceptable salt thereof. Pharmaceutically acceptable salts may include acid addition salts, and examples of acid addition salts include inorganic acids such as hydrochloric acid, hydrobromic acid, phosphonic acid and sulfuric acid and tartaric acid, acetic acid, citric acid, malic acid, maleic acid, which can be prepared by conventional methods. Salts of organic acids such as acids, methanesulfonic acid and gluconic acid are included, but are not limited thereto.

The preparation of the medicament of the present invention may be carried out according to methods known in the art, and generally known and used adjuvants and additional suitable carriers or diluents may be used. The pharmaceutical composition of the present invention may be used in the form of a solid agent, a solution, an emulsion, a dispersant, a micelle, a liposome, and the like, and the composition obtained therein may be used as an active ingredient with an organic or inorganic carrier or excipient suitable for oral or parenteral application. And the compounds of the present invention. The active ingredient is a pharmaceutically acceptable carrier that is generally non-toxic for, for example, tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, solutions, jellies and any other form suitable for use. Can be mixed together. Available carriers include solid, semi-solid or liquid glucose, lactose, gum arabic, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, urea, Medium chain length triglycerides, dextran and other carriers suitable for use in the preparation of formulations are included. In addition, auxiliary agents, stabilizers, thickeners, coloring agents and flavoring agents may be used. The white paper extract or imperatorin or a pharmaceutically acceptable salt thereof of the present invention is included in the pharmaceutical composition in an amount sufficient to exert a desirable effect on the progression of the disease or on the disease state. Pharmaceutical compositions containing the active ingredient are in a form suitable for oral use, e.g. tablets, oral tablets, lozenge tablets, solutions, jelly, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft It may be a capsule, or a syrup or elixir. Compositions for oral use may be prepared according to any method known in the art for the preparation of pharmaceutical compositions, and these compositions are excellent in pharmaceutically, such as sucrose, lactose, or saccharin to provide a formulation suitable for the mouth. It may contain one or more agents selected from the group consisting of sweetening agents, seasoning agents such as peppermint, hepatica or cherry, coloring agents and preservatives. Further, tablets containing the active ingredient together with non-toxic pharmaceutically acceptable excipients can be prepared by known methods. The excipients used are, for example, (1) inert excipients such as calcium carbonate, lactose, calcium phosphate or sodium phosphate; (2) granulating and disintegrating agents such as corn starch, potato starch or alginic acid; (3) a binder such as tragacanth rubber, corn starch, gelatin or acacia, and (4) a lubricant such as magnesium stearate, stearic acid or talc. Tablets may be uncoated or coated in a known manner to delay disintegration and absorption in the gastrointestinal tract so that the action can be maintained for a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be used. They can also be coated to form osmotic therapeutic tablets to control release.

In some cases, oral preparations may be used in the form of hard gelatin capsules in which the active ingredient is mixed with an inert solid-phase diluent such as calcium carbonate, calcium phosphate or kaolin. In addition, they may be in the form of soft gelatin capsules in which the active ingredient is mixed with water or an oily medium, for example peanut oil, liquid paraffin or olive oil.

The pharmaceutical composition may be in the form of a sterile injectable suspension. Such suspensions can be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. In addition, the sterile injectable preparation may be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solution, for example a solution in 1,3-butanediol. Sterile fixed oils are usually used as solvents or suspension media. For this purpose, synthetic mono- or diglycerides, fatty acids (including oleic acid), natural vegetable oils such as sesame oil, coconut oil, peanut oil, cottonseed oil, etc. or synthetic fat carriers such as ethyl oleate. Any non-irritating fixed oil can be used. If necessary, buffers, preservatives, antioxidants, and the like may be incorporated.

In addition, the compounds usable in the practice of the present invention may be administered in the form of suppositories suitable for rectal administration of drugs. Such compositions can be prepared by mixing the drug with suitable non-irritating excipients such as cocoa butter, synthetic glyceride esters of polyethylene glycol, which are solid at normal temperature but liquid and/or dissolve in the rectal cavity to release the drug.

Typical daily dosages of the white paper extract of the present invention or imperatorin or a pharmaceutically acceptable salt thereof are generally in the range of about 10 μg to about 200 mg/kg body weight, preferably in the range of about 50 μg to about 150 mg/kg body weight. Since each treatment target has varying degrees of symptoms and each drug has unique therapeutic properties, it is the task of the expert to determine the treatment target's response to treatment and to change the dosage accordingly.

In addition, the present invention provides a food additive or beverage composition for preventing or improving endoplasmic reticulum stress and/or mitochondrial function impairment containing white paper extract or imperatorin as an active ingredient.

When the white paper extract or imperatorin of the present invention is used as a food additive, each of them may be added as it is or may be appropriately used in accordance with a conventional method with other foods or food ingredients. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment). In general, white paper extract or imperitoline may be added in an amount of 0.0001 to 30% by weight, preferably 0.1 to 10% by weight, respectively, based on the raw material in the manufacture of food or beverage. Can be adjusted. There is no particular limitation on the type of the food. Examples of foods to which white paper extract or imperatorin can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, There are teas, drinks, alcoholic beverages, and vitamin complexes.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

Example

A low fat and high fat diet

The 10% Kcal low fat diet and 60% Kcal high fat diet used in the experiment were purchased from Research Diets (USA).

White paper extract and Imperatoline Produce

The white paper extract was used after extracting the white paper with 70% ethanol by cold needle at room temperature, and then dissolving the freeze-dried powder in water or a buffer solution. Imperatorin, one of the main effective ingredients of white paper, can be used by fractionating the methanol or ethanol extract of white paper with chlorform, separating and identifying it using silica gel and column chromatography. Lin was used and the purity was 99.16%.

Induction of endoplasmic reticulum stress

To induce endoplasmic reticulum stress, Tunicamycin, which is known to induce endoplasmic reticulum stress by inhibiting the glycosylation process (N-glycosylation) of newly synthesized proteins in the endoplasmic reticulum, or Tunicamycin, which is known to induce endoplasmic reticulum stress, is known to induce endoplasmic reticulum stress by destroying calcium homeostasis in the endoplasmic reticulum. Thapsigargin was used.

Reverse Transcriptase - Polymerase Chain Reaction ( RT - PCR )

^{Total RNA was extracted by adding 1 mL TRIzol ®} reagent (Invitrogen, USA) to a small amount of tissue or cells. The extracted RNA was electrophoresed on 1% agarose gel to confirm the bands of 18S and 28S, and the optical density was measured at wavelengths 260 nm and 280 nm to confirm that the ratio was within the range of 1.8-1.95, and the optical of each sample. RNA was quantified using the density value. 1µg RNA was converted to cDNA using Maxime RT Premix Kit (Intron Biotechnology, Korea), and PCR was performed with GeneAmp® PCR system 9700 (Applied Biosystems, USA) under optimal conditions using primers designed for each gene as follows. Was performed. And each PCR product was electrophoresed on 1% agarose gel, and the relative concentration was measured using an image densitometer (Alpha Ease FC software (Alpha Innotech, USA) on UV. The mRNA level was normalized by comparing it with the 18S rRNA level. The primers used for RT-PCR are as follows.

Oxygen respiration rate ( O ₂ consumption rate , OCR ) Measure

The muscle tissue collected from the sacrificed mice was 2.77 mM CaK ₂ EGTA, 7.23 mM K ₂ EGTA, 5.77 mM Na ₂ ATP, 6.56 mM MgCl ₂ 6H ₂ O, 20 mM Taurine, 15 mM Na ₂ Phospho-creatine, 20 mM Placed in a cold BIOPS solution (pH 7.1) containing Imidazole, 0.5 mM Dithiothreitol, 50 mM MES, and 50 μg/mL saponin, and stirred for 30 minutes. The muscle tissue in the BIOPS solution contains 20 mM HEPES, 110 mM sucrose, 3 mM MgCl ₂ 6H ₂ O, 0.5 mM EGTA, 10 mM KH ₂ PO ₄ , 60 mM K-lactobionate, 20 mM Taurine, 0.1% BSA. Transferred to cold respiration buffer (pH 7.1) and stirred for 10 minutes to increase permeability of the cell membrane. After moving the muscle tissue with increased cell membrane permeability to the Oxygraph-2k chamber (Oroboros, Austria) maintained at 37°C, 100 mM p1, p5-di(adenosine-5')-pentaphosphate was injected into the chamber. It was prevented from becoming adenylate. OCR measured the basal respiration rate of each tissue, and then measured oxygen consumption (OCR) by sequentially adding substrates and inhibitors involved in each OXPHOS complex (complex I, II & III, IV). Substrates and inhibitors used to measure each OXPHOS complex include 2 mM ADP, 8 mM malate, 20 mM glutamatez (Complex I), 1 mM rotenone, 10 mM succinate (Complex II & III), 25 mM antimycin A, 80 μM ascorbate, 420 μM N,N,N',N'-tetramethyl-P-phenylenediamine dihydrochloride (TMPD) [Complex IV] was used. The measured OCR was expressed as pmoles/sec/mg wt.

<Experimental Example 1> White paper extract against endoplasmic reticulum stress or Imperatoline effect

One) Hep G2 Effects on apoptosis by endoplasmic reticulum stress

Hep G2 cells in a solution containing 10% fetal bovine serum (FBS) in a minimum essential medium (MEM) at 37°C, 5% carbon dioxide (CO ₂ ), and 95% air (O ₂ ). Cultured. White paper extract (ADEx, 0.5 μg/mL) or imperatoline (IMP, 0.1 μg/mL) was added to Hep G2 cells (1×10 ^{4 cells/well) in culture, followed by pretreatment for 5 hours.} Toxigagin (3 μg/mL) or tunicamycin (4 μg/mL), which induces endoplasmic reticulum stress, was added to each of the white paper extract or imperatorin-treated and non-treated groups, and cultured for 24 hours to recover cell death. The effect was measured by MTT assay. In terms of cell viability compared to the control group (DMSO treated group) that did not treat both white paper extract and thapsigagin or tunicamycin, the white paper extract or imperatorin pretreatment group almost completely protects the death of hepatocytes induced by endoplasmic reticulum stress. It could be observed that (Fig. 1).

2) Hep G2 Endoplasmic reticulum stress in cells Marker Effects on gene expression

Hep G2 cells in a solution containing 10% fetal bovine serum (FBS) in a minimum essential medium (MEM) at 37°C, 5% carbon dioxide (CO ₂ ), and 95% air (O ₂ ). Cultured. White paper extract (ADEx; 0.5 μg/mL) or imperatorin (IMP; 0.1 μg/mL) was added to Hep G2 cells (1×10 ^{6 cells/well) in culture, followed by pretreatment for 5 hours.} Thapsigagin (3 μg/mL), which induces endoplasmic reticulum stress, was added to the white paper extract or imperatorin-treated and untreated groups, and cultured for 24 hours. RNA was isolated from each test group and the expression of endoplasmic reticulum stress marker genes GRP78 and XBP1p (processed form of XBP1) was observed through RT-PCR. As shown in FIGS. 2 and 3, expression of endoplasmic reticulum stress marker genes GRP78 and XBP1p was reduced in the group treated with white paper extract or imperatorin than in the group treated with tapsigagin (Thap group).

3) Hep G2 Endoplasmic reticulum stress in cells ATP Effect on reduction in quantity

Hep G2 cells in a solution containing 10% fetal bovine serum (FBS) in a minimum essential medium (MEM) at 37°C, 5% carbon dioxide (CO ₂ ), and 95% air (O ₂ ). Cultured. Thap; 3 μg/mL, which induces endoplasmic reticulum stress after 5 hours of pretreatment by adding imperatoline (IMP; 0.1 μg/mL) to cultured Hep G2 cells (1×10 ^{5 cells/well) and incubating} ) Or tunicamycin (Tuni; 4 μg/mL) was added and incubated for 24 hours (Thap+IMP group or Tuni+IMP group in FIG. 4). However, imperitoline was not added to the control group (DMSO group, Thap group, or Tuni group). ATP concentration was measured in all groups of cells (1×10 ⁵ cells/mL) using an ATP-Bioluminescence assay kit (#FLASC; Sigma-Aldrich, USA).

As shown in FIG. 4, in the IMP group to which only imperatoline was added, the ATP concentration tended to slightly increase compared to the control group, but did not show a significant difference, and in the endoplasmic reticulum stress (Thap group, Tuni group), the ATP concentration was It was found that it decreased significantly. However, when tapsigagin or tunicamycin and imperatorin were treated together, the ATP concentration was significantly increased compared to the Thap group and the Tuni group. These results are thought to be due to the fact that imperatorin increased mitochondrial activity by alleviating both typical endoplasmic reticulum stresses, thereby increasing ATP production.

<Experimental Example 2> Effect of white paper extract in diabetic model animals

Six-week-old Lean (C57BL/6J) and leptin receptor-modified C57BL/6J db/db (SLC, Japan) male mice were used. Lean and db/db mice, which are diabetic model animals, were divided into 2 groups, a control group and an experimental group, respectively, water (CTL) was administered to the control group, and white paper extract (ADEx) was administered to the experimental group, and a total of 4 groups were divided. Each group used 4 mice. All experimental animals were fed a chaw diet freely for 33 days. The experimental group was administered with a dose of 100 mg/kg/day of white paper extract once a day using oral-zonde during the experiment period, and the control group was administered the same amount of water in the same manner.

1) weight change

Although there was no change in dietary intake of each group during the experiment, as a result of measuring body weight by day, lean mice showed little change in body weight by administration of white paper extract, but showed about 1.4 times higher body weight than lean mice. In db/db mice, the amount of weight gain decreased when the white paper extract was administered (Fig. 5).

2) white fat ( epididymal fat ) For

After the administration period of the white paper extract was over, the white fat part of the testis was separated from the sacrificed mice and the weight was measured. Administration of white paper extract (ADEx) significantly reduced the amount of testis white fat in db/db mice, and there was a tendency to decrease in lean mice, but it was not statistically significant (Fig. 6).

3) Effect on mitochondrial activity

After the administration period of the white paper extract was over, the mitochondria were isolated from the soleus muscle of the sacrificed mice (Choi et al, 2005; Gogvadze et al., 2004), and the results of measuring the amount of oxygen consumption (oxygen respiration rate) were shown. It is shown in 7. Administration of white paper extract (ADEx) increased mitochondrial activity of soleus muscle in mice in both lean mice (lean group) and db/db mice (db/db group). In particular, the administration of imperatorin increased the oxidative phosphorylation (OXPHOS) complex to a remarkably higher level in the case of disease (db/db group) than in the normal state.

4) Effects on blood sugar and insulin resistance

In order to measure blood glucose or insulin resistance, the test animal was kept on an empty stomach for 16 hours, and then 2 g/kg glucose was administered orally to measure blood glucose by hour (0, 15, 30, 60, 120 minutes). Tolerence test, OGTT oral glucose tolerance test) was performed (Fig. 8).

The db/db group showed about 4-5 times higher blood sugar than the control group, but when the white paper extract (ADEx) was administered, the insulin sensitivity was improved by imperatoline, and the blood sugar decreased in a short time compared to the db/db group. I did. These results were caused by the administration of imperatoline for 33 days, and if imperatorin was administered for a long period of time, better results could be obtained. In addition, although it was administered for a short period of time, it was thought that the initial insulin sensitivity increased, so it was thought that it had a good effect on the insulin secretion function of the pancreas.

<Experimental Example 3> Intake of a high fat diet In mice Imperatoline effect

Six-week-old C57BL/6J (SLC, Japan) male mice were used as a control group in a low-fat diet group (LF; #D12450B; Research diets, USA; 10% Kcal fat), a high-fat diet group (HF; #D12492; Research). diets; 60%Kcal fat), a group treated with 5 mg/kg/day or 10 mg/kg/day of imperatoline (IMP) with a high-fat diet (HF+IMP5 and HF+IMP10), a total of 4 Divided into groups. Each group used 7 mice. The experimental animals were fed a low-fat diet and a high-fat diet for 7 weeks, respectively, and the group treated with imperitoline was administered imperatorin through oral administration using oral-zonde during the experimental period. The experimental animals bred as described above were sacrificed 7 weeks later, and blood, brain, hypothalamus, liver, and soleus muscle were extracted and tested.

1) Changes in body weight and dietary intake

Table 1 below shows the change in average weight of each experimental animal group, and Tables 2 and 9 show the results of measuring the change in dietary intake rate.

In the experimental animals, the body weight and dietary intake rate (kcal/day) were increased during the experiment period, and the body weight and dietary intake tended to decrease by administration of imperatorin (IMP).

2) Effect on the expression of an appetite-regulating neuropeptide in the hypothalamus

In order to find out the cause of the decrease in dietary intake, the hypothalamus was isolated from mice in each group, and the expression of neuropeptide mRNA involved in appetite regulation was measured by RT-PCR. It was observed that the mRNA of NPY (neuropeptide Y) and AgRP (agouti-related protein), which are appetite-enhancing neuropeptides, was increased due to intake of a high fat diet, and their expression was decreased due to administration of imperatorin (IMP) (Fig. . Therefore, imperatorin appears to exhibit an appetite suppressing effect by inhibiting the expression of NPY and AgRP.

3) white fat ( epididymal fat ) For

Consumption of a high fat diet results in weight gain and excessive fat accumulation. White fat (epididymal fat) of the experimental animal group from the testis After collection, the weight was measured. The accumulation of excessive fat due to the intake of a high fat diet significantly decreased white fat at high concentrations when imperatorin (IMP) was administered together (FIG. 11). These results show that imperatorin is effective in reducing body fat accumulated by a high fat diet.

4) Effects on fatty liver

Excessive intake of fat causes the intake of fat beyond its ability to decompose in the liver, forming fatty liver, where fat is no longer decomposed and fat accumulates in the liver. When the fatty liver becomes more severe, liver fibrosis progresses and eventually the liver cannot function properly. Therefore, in order to see the morphology of liver tissue cells when ingesting a high-fat diet, it was confirmed by immunohistochemistry and shown in FIGS. 12 and 13.

12 is a result of confirming the expression of C/EBP-α, a protein that plays a major role in fat metabolism and adipocyte differentiation. It can be seen that C/EBP-α, which is excessively expressed due to the intake of a high fat diet, is normalized to a level similar to that of the control group when imperatorin is administered.

In addition, Figure 13 is a result of confirming the morphology of hepatocytes through H&E staining. In the control group, the shape of the cell was shown as it was, but in the group that consumed the high fat diet, the shape of the cell was damaged so much that it was impossible to recognize it, and it was confirmed that many lipid droplets were generated. It was found that when imperatorin was administered, it was normalized to a form of hepatocyte similar to that of the control group in a dose-dependent manner.

This result shows that imperatorin inhibits the synthesis or accumulation of fat in the liver due to ingestion of a high fat diet and has excellent efficacy in restoring liver function.

5) Effect on mitochondrial activity

The mitochondrial oxygen respiration rate was measured using the soleus muscle isolated from the mice to be tested. FIG. 14 shows that although a high fat diet did not significantly reduce the amount of mitochondrial oxygen consumption, administration of imperatorin improves mitochondrial activity of muscles, particularly the activity of OXPHOS complex I.

6) oral Glucose tolerance exam ( OGTT , oral glucose tolerance test )

In order to measure insulin resistance, the experimental animals were kept in a fasting state for 16 hours, and then 2 g/kg glucose was orally administered to measure blood glucose by time (0, 15, 30, 60, 120 minutes), and the results are shown in FIG. 15. In the high fat diet group, compared to the normal diet group, fasting blood sugar increased, and high blood sugar continued 2 hours after glucose administration, resulting in diabetes and insulin resistance. On the other hand, in the high-fat diet group administered with imperitoline, fasting blood sugar slightly decreased and the blood glucose removal rate increased after glucose intake, but the blood sugar was not completely normalized. These results show that imperatorin does not completely normalize the increase in blood sugar and insulin resistance caused by a high fat diet, but can improve insulin resistance.

<Production Example>

It is possible to make a product that can be administered through various routes as follows, including the white paper extract or imperatorin of the present invention, but is not limited thereto.

Capsule

The ingredients of white paper extract 20.0% (w/w) / lactose 79.5% (w/w) / magnesium stearate 0.5% (w/w) are mixed and dispersed into capsules containing about 100 mg each.

refine

Imperatorin 20.0% (w/w) / Magnesium stearate 0.5% (w/w)

Croscarmellose sodium 2.0% (w/w) / lactose 76.5% (w/w)

PVP (polyvinylpyrrolidine) 1.0% (w/w)

The ingredients are combined and granulated using a solvent such as methanol. Thereafter, the formulation is dried and tablets are formed with an appropriate tablet press.

beverage

9% by weight of white paper extract, 10% by weight of fructose, 1% by weight of refined sugar, 0.2% by weight of sodium citrate were mixed with purified water to a total of 100% by weight and stirred to prepare a beverage according to a conventional beverage preparation method.

references

1. Beal, M. F. (1995). Mitochondrial dysfunction and oxidative damage in neurodegenerative diseases. Austin: R.G. Landes Co.

2. Banhegyi, G., Baumeister, P., Benedetti, A., Dong, D., Fu, Y., Lee, AS, et al. (2007). Endoplasmic reticulum stress. Ann NY Acad Sci , 1113, 58-71.

3. Chen, JX & Yan, SD (2007). Amyloid-beta-induced mitochondrial dysfunction. J Alzheimers Dis , 12(2), 177-184.

4. Champe, P. C., Harvey, R. A., & Ferrier, D. R. (2005). Biochemistry (3rd ed). Philadelphia: Lippincott/Williams & Wilkins.

5. Choi YS, Ryu BK, Min HK, Lee SW, and Pak YK (2005) Analysis of proteome bound to D-loop region of mitochondrial DNA by DNA-linked affinity chromatography and reverse-phase liquid chromatography/tandem mass spectrometry, Ann . NY Acad . Sci . 1042. 88-100

6. Gogvadze, V., Orrenius, S., & Zhivotovsky, B. (2004). Assessment of celll toxicity. In M. D. Maines (Ed.), Current protocols in toxicology (pp. 2.10.13-2.10.14). New York: John Wiley.

7. Jeffery, RW, & French, SA (1997). Preventing weight gain in adults: design, methods and one year results from the Pound of Prevention study. Int J Obes Relat Metab Disord , 21(6), 457-464.

8. Lindholm, D., Wootz, H., & Korhonen, L. (2006). ER stress and neurodegenerative diseases. Cell Death Differ , 13(3), 385-392.

9. Must, A., Spadano, J., Coakley, EH, Field, AE, Colditz, G., & Dietz, WH (1999). The disease burden associated with overweight and obesity. JAMA , 282(16), 1523-1529.

10. National Institutes of Health (2004). Insulin resistance and pre-diabetes. MD, USA: Author

11. Scheffler, I. E. (2008). Mitochondria (2nd ed.). Hoboken, N. J.: Wiley-Liss.

12. Tsiotra, PC, & Tsigos, C. (2006). Stress, the endoplasmic reticulum, and insulin resistance. Ann NY Acad Sci , 1083, 63-76.

13. Wallace, DC (2005). A mitochondrial paradigm of metabolic and degenerative diseases, aging, and cancer: a dawn for evolutionary medicine. Annu Rev Genet , 39, 359-407.

14. World Health Organization (2004). Obesity: preventing and managing the global epidemic Report of a WHO Consultation. Geneva, Switzerland: Author

15. Wolf, AM, & Colditz, GA (1998). Current estimates of the economic cost of obesity in the United States. Obes Res , 6(2), 97-106.

16. Yoshida, H. (2007). ER stress and diseases. FEBS J , 274(3), 630-658.

17. Zhao, L., & Ackerman, SL (2006). Endoplasmic reticulum stress in health and disease. Curr Opin Cell Biol , 18(4), 444-452.

18. Jaeheon Kang, Namsoon Kim. (2002) Obesity trends in Korea. Journal of the Korean Society of Obesity , 11(4), 329-336.

19. Mi-Kyung Kim, Geun-Gyu Park (2008). Endoplasmic Reticulum Stres and Diabetes. Journal of the Korean Endocrinology Society , 23(1), 1-8.

20. Kim Young-seol. (1990). Classification and evaluation of obesity. Journal of the Korean Nutrition Society , 23(5), 337-340.

21. Kim Jong-gyun, Lim Jeong-su, Lim Jun, Park Sang-hyun, Hong Doo-ho. (2007). The difference in youth obesity in Incheon according to economic level. Journal of the Korean Society of Obesity . 16(2), 76-85.

22. Yunna Lee, Haejong Shin, Bokhee Kim, Youngae Jang, Choil Kim. (2004). Dietary characteristics of the obese population in Korea. Korean Society for Obesity Spring Conference

23. Choi Hye-mi, Seo Yeon-kyung. (2004). “Obesity and metabolic syndrome”-How is their diet? Korean Society for Obesity Spring Conference

<110> UNIVERSITY-INDUSTRY COOPERATION GROUP OF KYUNG HEE UNIVERSITY <120> Composition for ameliorating the ER stress or the mitochondrial dysfunction comprising the extract of Angelica dahurica or imperatorin <130> PA120418/KR-DIV-1 <160> 10 <170> KopatentIn 2.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer (F) <400> 1 gagatcatcg ccaacgatca g 21 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer (R) <400> 2 acttgatgtc ctgctgcaca g 21 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer (F) <400> 3 ggtctgctga gtccgcagca gg 22 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer (R) <400> 4 gggcttggta tatatgtgg 19 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer (F) <400> 5 gcttgaagac ccttccatgt ggtg 24 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer (R) <400> 6 ggcggagtcc agcctagtgg 20 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer (F) <400> 7 gaaggcctga ccaggctctg ttcc 24 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer(R) <400> 8 aaaggcattg aagaagcggc ag 22 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer (F) <400> 9 gagcgaaagc atttgccaag 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer(R) <400> 10 ggcatcgttt atggtcggaa 20

Claims (5)

Blank ( Angelica group consisting of obesity, body fat increase, lipid metabolism, fatty liver, liver fibrosis, liver disease, pancreatic disease, metabolic syndrome, hypertension, arteriosclerosis, cardiovascular disease, cancer and aging, including dahurica ) extract and pharmaceutically acceptable carrier Pharmaceutical composition for the prophylaxis or treatment of a disease selected from. Obesity, increased body fat, lipid metabolism, fatty liver, liver fibrosis, liver disease, pancreatic disease, metabolic syndrome, hypertension, arteries, including imperatorin or its pharmaceutically acceptable salts and pharmaceutically acceptable carriers A pharmaceutical composition for the prevention or treatment of a disease selected from the group consisting of sclerosis, cardiovascular disease, cancer and aging. The pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutical composition is characterized by alleviating vesicle stress and / or improving mitochondrial function. Tablets, tablets, lozenges, liquids, jellies, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs comprising the pharmaceutical composition of claim 1 or 2 pharmaceutical preparations selected from the group consisting of (elixir). Blank ( Angelica obesity, body fat increase, lipid metabolism, fatty liver, liver fibrosis, liver disease, pancreatic disease, metabolic syndrome, hypertension, arteriosclerosis, cardiovascular disease, cancer, and the like, including dahurica ) extract or imperatorin (imperatorin) as an active ingredient A food composition for preventing or ameliorating a disease selected from the group consisting of aging.

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