KR20100060279A - Composition for ameliorating the er stress or the mitochondrial dysfunction comprising the extract of angelica dahurica or imperatorin - Google Patents

Abstract

PURPOSE: A pharmaceutical containing Angelica dahurica extract or imperatorin as an active ingredient is provided to prevent or treat diseases caused by endoplasmic reticulum stress or mitochondria malfunctions. CONSTITUTION: A medicinal composition for preventing or treating diseases caused by endoplasmic reticulum stress or mitochondria malfunction contains Angelica dahurica extract or imperatorin. The diseases are liver fibrosis, metabolic syndrome, hypertension, neurodegenerative disorder, bipolar disorder, diabetes, artery scleroma, and ischemia. A medicinal formulation contains the medicinal composition and is used in the form of a tablet, oral tablet, liquids, jelly, aqueous or oil suspension, dispersion powders, or granules, emulsion, soft or hard capsule, or syrup.

Description

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

The present invention relates to a pharmaceutical composition or a food composition useful for improving ER stress or dysfunction of mitochondria containing white paper ( Angelica dahurica ) extract or imperatorin (imperatorin) as an active ingredient.

The endoplasmic reticulum contains rough ER (RER) and smooth ER (SER) with ribosomes attached. Rough endoplasmic reticulum is the main function of protein synthesis, and about one third of the protein in the cell is synthesized in the rough endoplasmic reticulum. But vesicular vesicles are found in most cells and play an important role in synthesizing various lipids and steroid hormones and regulating intracellular calcium levels with calcium reservoirs. Due to various physiological or pathological conditions, protein folding does not occur properly when calcium homeostasis, glycosylation of glycoproteins, or disulfide bonds are formed in the endoplasmic reticulum. When the endoplasmic reticulum accumulates more than the unfolded protein or calcium depletion, this condition is called endoplasmic reticulum stress (Zhao & Ackerman, 2006; Kim Mi-kyung and Park Geun-gyu, 2008). ER stress causes cellular damage by increasing apoptosis and the formation of large amounts of reactive oxygen species (ROS) through multiple signaling.

Diseases caused by vesicle stress include neurorodegenerative disease, bipolar disorder, diabetes mellitus, atherosclerosis, inflammation, ischemia, heart disease (heart) It is known to be involved in the development of various diseases such as diseases, liver diseases (kidney diseases), cancer (cancer), etc., and in recent years have been reported about the relationship between obesity, diabetes. These findings suggest that endoplasmic reticulum stress relief may enable the prevention / treatment of metabolic syndrome including diabetes (Banhegyi et al., 2007; Lindholm et al., 2006; Tsiotra & Tsigos, 2006; Yoshida, 2007).

In general, mitochondria are organelles within cells that are known as "cell power stations." Mitochondria not only produce ATP in the cell, but also play essential functions in cell / object life, including apoptosis, ion homeostasis, sugar and fat metabolism, and pyrimidine / urea / heme synthesis. do.

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

Past genetic studies of mitochondria associated with human disease have demonstrated that mutations in mitochondrial genes cause metabolic and neurological genetic diseases (Wallace, 2005). Recent studies reported by various laboratories, including those of the inventors of the present invention, have shown that dysfunction of mitochondria due to genetic and environmental causes is due to metabolic syndrome (obesity, diabetes, lipid metabolism, hypertension). The above symptoms have been shown to be simultaneously associated with a single person and are directly linked to chronic inflammatory diseases, including neurological disease, neurodegenerative diseases, and aging, including insulin resistance. For example, lean body mass and fat mass are correlated with insulin resistance in elderly people, and mitochondrial oxidative phosphorylation activity of the elderly was measured by in vivo NMR technique. This decrease was about 40%, suggesting that mitochondrial dysfunction is a causative agent of insulin resistance (Pettersen et al., 2003). In addition, it has been reported that PGC-1α, a co-activator that enhances the activity of ligands of peroxisome proliferators activated receptor γ (PPARγ), which is used as a new therapeutic agent for insulin resistance, promotes the production of mitochondria. Therefore, it is accepted that a clear causal relationship is established between the function of mitochondria and the development of insulin resistance and metabolic syndrome.

Overweight and obesity is a condition that increases the percentage of fatty tissues in the body that is excessively over the body, which is not only a risk factor for various diseases, but also a health risk factor. Active management is needed. Although it is a single disease, it is a direct and indirect cause of various serious health problems. Increasing the diet and westernization of modern people's busy life increases the intake of foods high in calories, high in fat and salt, while the decrease in activity increases the frequency of obesity (Kim Jong-kyun, 2007; Et al., 2004). As a result, socio-economic losses are on the rise (Wolf & Colditz, 1998). According to the domestic epidemiological survey, obesity among Koreans is also reported to be more than double the prevalence of obesity among low-income and low-educated underprivileged people, as in the West, and increasing various chronic diseases. Recent studies have reported that overweight and obesity have a high burden of diseases such as diabetes, ischemic heart disease, and stroke. In particular, Asians have lower obesity standards than foreign countries. In Korea, overweight problems are more serious than foreign obesity (Jeffery & French, 1997; Must et al., 1999; And Kim Nam-soon, 2002; Kim Young-sul, 1990; Choi Hye-mi et al., 2004).

When both parents are obese in a low-calorie, high-calorie environment, their eating habits are passed on to their children, resulting in 70-80% of their children becoming obese. In addition, in the case of adult obesity, the size of fat cells increases, but in the case of child obesity, the number and size of fat cells increases, obesity of children is more dangerous than adult obesity, and various diseases including the incidence of adult disease when obese children become adults Increasing risk of urine is an urgent need for prevention or treatment (Champ et al., 2005). As such, overweight and obesity are serious problems that require socially active management beyond personal problems. Recently, the World Health Organization (WHO) recognizes that obesity, which spreads like an epidemic, is a big problem that can be defined as a disease (World Health Organization, 2004).

Diabetes is largely classified into type 1 and type 2 diabetes. Type 1 diabetes occurs in children and puberty, and diabetes is caused by deficiency of insulin due to destructive lesions of the β cells of the pancreas. Type 2 diabetes occurs after age 35 and is caused by a decrease in insulin secretion and insulin resistance (cell resistance to insulin) that insulin-sensitive peripheral tissues such as liver, muscle, adipocytes and pancreas do not respond to insulin. The disease results in a decrease in glucose tolerance to maintain blood sugar levels despite food intake due to insulin deficiency or insulin resistance, leading to high blood sugar levels and long-term diabetes, which causes glucose to be excreted in the urine. Symptoms of early diabetes are weight loss due to poor ability to maintain urination, thirst and blood sugar, resulting in increased appetite and increased insulin resistance (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 been reported so far, and most of them are drugs aimed at alleviating symptoms. Troglitazone recently used as a drug for insulin resistance PPARγ ligands of the lineage are still limited in use due to side effects such as secondary obesity and edema that may be observed in Koreans due to low therapeutic rates and properties that promote adipogenesis. Therefore, if a drug is developed that can prevent / recover endoplasmic reticulum stress and / or mitochondrial impairment, which has low side effects and is considered to be a cause of diabetes / obesity / neurodegenerative disease, environmental or genetic factors It could be developed as a therapeutic agent to prevent / treat metabolic syndrome and related degenerative diseases caused by These drug candidates should be excellent in reducing vesicle stress and improving mitochondrial function, and should also have an effect of reducing appetite, unnecessary body fat and improving insulin resistance. However, no substance has been reported that still has this effect.

On the other hand, Angelica dahurica is a 2-3 year-old herb native to Korea, China, and Japan. In Chinese medicine, the herbal medicine made by drying the root is called “white paper.” The main bioactive substance is imperitorin, It has been reported as isoimperatorin, oxypeucedanin, pelletopterin, and byakangelicol. In traditional Korean medicine, the efficacy of various white papers is known, especially for sweating, sedation, pain, colds, headaches, toothaches, whitening, pore shrinkage, anti-inflammatory action is known. However, there is no known effect on the regulation of vesicle stress or mitochondrial function or the effects related to insulin resistance, obesity, diabetes and fatty liver.

The pharmaceutical composition containing white paper extract and imperitorin as an active ingredient is disclosed, for example, in Korean Patent Registration No. 10-0527876, “convulsability containing imperitorin and / or falcarindaiol as an active ingredient. A pharmaceutical composition aimed at the anti-convulsant treatment and prevention of impairtorin and / or falcarindaiol, which is distributed and extracted from copper roots, with low side effects under the name of the composition for the prevention and treatment of diseases. , And other Korean patent registration No. 10-036152 proposes a "white paper, Gobon extract and a cosmetic composition containing the extract." However, the composition is to prevent the development of fatty liver and metabolic syndrome through the improvement of mitochondrial function Inhibition or reduction slows the development of metabolic syndrome and improves symptoms of obesity, diabetes and aging Key Bar is not shown at all for efficacy.

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

Therapeutics for diseases such as insulin resistance, obesity, diabetes and fatty liver are most often alleviated 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 impairment, which is indicated as a cause of the disease.

In order to solve the above problems, the inventors of the present invention have found that for the first time that surprisingly natural white paper extract and imperitorin alleviate or improve the endoplasmic reticulum stress and mitochondrial function impairment, and provide it as a preventive or therapeutic agent for related diseases. do.

The composition containing the white paper extract or imperitorin (imperatorin) of the present invention as an active ingredient has an effect of relieving vesicle 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 stress relief of the endoplasmic reticulum and / or improve the function of the mitochondria.

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

Therefore, the pharmaceutical composition of the present invention is obesity, insulin resistance, body fat increase, fatty liver, liver fibrosis, metabolic syndrome, lipid metabolism abnormality, hypertension, neurodegeneration associated with stress relief and / or mitochondrial dysfunction of the endoplasmic reticulum It can be used for the prevention or treatment of diseases, bipolar 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.

The present invention relates to a pharmaceutical composition or a food composition for reducing stress and / or improving the function of the mitochondria of the endoplasmic reticulum containing the white paper extract or imperatorin (imperatorin) as an active ingredient.

The present invention also provides methods and kits for the use of white paper extracts or imperitorins to relieve stress in the endoplasmic reticulum and / or to improve the function of mitochondria.

The inventors of the present invention induced the endoplasmic reticulum stress in Hep G2 cells, but apoptosis is reduced when pre-treated with white paper extract (Fig. 1), the expression of the vesicle stress marker gene by the blank extract or imperitorin It was found to decrease (Figures 2 and 3). It was also observed that inducing endoplasmic reticulum stress in Hep G2 cells decreases the amount of intracellular ATP but significantly increases the amount of ATP when treated with imperitorin (FIG. 4). This may be due to increased ATP production by increasing mitochondrial activity.

In addition, the inventors of the present invention administered the white paper extract (ADEx) to diabetic model mice (db / db mice) and lean mice for 33 days and observed the weight change, even though no change in the dietary intake of each group was observed. In the case of lean mice, the weight gain was not affected by the white paper extract, but in db / db mice, the weight gain decreased when the blank extract was administered (FIG. 5). In addition, the white fat content of the testis was significantly reduced in the db / db mice administration of white paper extract (Fig. 6). Administration of white paper extract increased mitochondrial activity of the soleus muscle of mice in both lean and db / db mice. In particular, administration of white paper extract significantly increased the activity of oxidative phosphorylation (OXPHOS) complexes in disease-prone (db / db mice) than normal conditions (Figure 7). In the oral glucose tolerance test, the db / db mice showed about 4-5 times higher blood glucose than the control group, but when the extract was administered, the insulin sensitivity was enhanced by the extract, which was observed to decrease the blood glucose level than the db / db group (Fig. 8).

The inventors of the present invention found that high-fat diet mice increased body weight and dietary intake as compared to low-fat diet mice, but weight gain and dietary intake were decreased when imperitorin (IMP) was administered (Tables 1 and 2, 9). The decrease in dietary intake increased mRNAs of the hypothalamic appetite-promoting neuropeptides NPY (neuropeptide Y) and AgRP (agouti-related protein) due to high-fat dietary intake, and these expressions were reduced by imperitorin administration. As shown in Fig. 10, imperitorin appears to exhibit an appetite suppressing effect by inhibiting the expression of NPY and AgRP. In addition, the inventors found that the fat accumulated excessively with weight gain due to the high fat diet was significantly reduced at high concentrations when impairatorin was administered together (FIG. 11). 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 inventors confirmed by immunohistochemistry, the expression of C / EBP-α overexpressed due to high-fat diet was impaired upon administration of imperitorin. Normalized to a level similar to the control (Fig. 12), the lipid lipids of the high-fat diet group normalized to a hepatocellular form similar to the control when dosed with imperitorin (Fig. 13). These results show that impairitorin has an excellent effect on restoring liver function by inhibiting synthesis or accumulation of fat in the liver due to high fat diet. In addition, the administration of imperitorin in high-fat diet group improved the mitochondrial activity of muscle, especially the activity of OXPHOS complex I (Fig. 14), slightly fasting blood glucose and the increase in blood glucose removal rate after glucose intake Observation was made (FIG. 15).

therefore. White paper extract or imperitorin of the present invention 1) reduce the risk of obesity by reducing the increased weight and appetite due to genetic or environmental factors. 2) It improves mitochondrial function through relieving vesicle stress, which strongly improves insulin resistance, helping to absorb glucose in blood into each tissue in a short time. 3) By reducing the weight of white adipose tissue to suppress unnecessary body fat increase to inhibit or reduce the accumulation of fat in each tissue. And 4) inhibit or reduce fatty liver or hepatic fibrosis. Therefore, if the composition containing the white paper extract and imperitorin according to the present invention exhibits a potent action that can prevent various diseases that can be caused by irregular eating habits and insufficient exercise amount, preventing and treating metabolic syndrome and accompanying fatty liver. As a pharmaceutical composition.

In addition, the white paper extract or imperitorin of the present invention can be used for the prevention, improvement or treatment of diseases caused by endoplasmic reticulum stress or impaired function of the mitochondria. Preventable or treatable diseases include, for example, obesity, insulin resistance, body fat gain, fatty liver, liver fibrosis, metabolic syndrome, lipid metabolism, hypertension, neurodegenerative disorders, 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.

"Treatment" in the present invention means the inhibition and alleviation of the progression of the disease. Terms not otherwise defined herein have the meanings commonly used in the art.

In the present invention, the white paper extract and imperitorin can be obtained by dividing and extracting organic solvent (for example, but not limited to n-butanol, methanol, ethanol, chloroform, and the like) and water from the root of white paper. There is .

Imperitorin 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 and tartaric acid, acetic acid, citric acid, malic acid, hydrochloric acid, hydrobromic acid, phosphonic acid, and sulfuric acid, which may be prepared by conventional methods. Salts of organic acids such as maleic acid, methanesulfonic acid and gluconic acid are included, but are not limited to these.

The preparation of a medicament of the present invention can be carried out according to methods known in the art, and commonly known and used auxiliaries and further suitable carriers or diluents can be used. The pharmaceutical compositions of the present invention may be used in the form of solids, solutions, emulsions, dispersants, micelles, liposomes and the like, wherein the composition obtained is used as an active ingredient with an organic or inorganic carrier or excipient suitable for oral or parenteral application. It includes a compound of the present invention. The active ingredient may be, for example, a pharmaceutically acceptable carrier which is generally nontoxic to tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, solutions, jelly and any other form suitable for use. Can be mixed together. Carriers that can be used include solid, semisolid or liquid glucose, lactose, gum arabic, gelatin, mannitol, starch paste, magnesium trisilicate salt, talc, corn starch, keratin, colloidal silica, potato starch, urea, Triglycerides, dextran, which have a medium chain length, and other carriers suitable for use in the preparation of the formulations are included. In addition, auxiliaries, stabilizers, thickeners, colorants and flavoring agents can be used. The blank extract or imperitorin or pharmaceutically acceptable salt thereof of the present invention is included in the pharmaceutical composition in an amount sufficient to have a desirable effect on disease progression or disease state. Pharmaceutical compositions comprising the active ingredient may be in a form suitable for oral use, eg, tablets, tablets, lozenges, solutions, jelly, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft Capsules, or syrups or elixirs. Compositions used for oral use may be prepared according to any method known in the art for the manufacture of pharmaceutical compositions, which compositions may be prepared such as sucrose, lactose or saccharin in order to provide a pharmaceutical pharmaceutically good and fit formulation. One or more agents selected from the group consisting of sweetening agents, mints, seasonings such as peppermint grass or cherry, colorants and preservatives. In addition, tablets comprising the active ingredient in combination with nontoxic pharmaceutically acceptable excipients may be prepared by known methods. Excipients used include, 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) binders such as tragacanth gum, corn starch, gelatin or acacia, and (4) lubricants such as magnesium stearate, stearic acid or talc. Tablets may be uncoated or coated in a known manner to delay degradation and absorption in the gastrointestinal tract so that the action can be maintained for a longer period of time. For example, a time delay material such as glyceryl monostearate or glyceryl distearate can be used. They may also be coated to form osmotic therapeutic tablets for controlling release.

In some cases, oral formulations 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. They may also be in the form of soft gelatin capsules in which the active ingredient is mixed with water or an oily medium such as peanut oil, liquid paraffin or olive oil.

The pharmaceutical composition may be in the form of a sterile injectable suspension. Such suspensions may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. In addition, sterile injectable preparations may be sterile injectable solutions or suspensions in nontoxic parenterally acceptable diluents or solutions, for example solutions in 1,3-butanediol. Sterile fixed oils are conventionally employed as a solvent or suspending medium. 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 and the like, or synthetic fat carriers such as ethyl oleate, etc. Any non-irritating fixed oil may be used. If necessary, buffers, preservatives, antioxidants, and the like may be incorporated.

In addition, compounds which may be used in the practice of the present invention may be administered in the form of suppositories suitable for rectal administration of the medicament. Such compositions may be prepared by mixing the agent with a suitable non-irritating excipient such as cocoa butter, synthetic glyceride esters of polyethylene glycol, which are solid in normal temperature but liquefied and / or dissolved in the rectal cavity to release the drug.

Typical daily dosages of the white paper extract or imperitorin or pharmaceutically acceptable salts thereof of the present invention generally range from about 10 μg to about 200 mg / kg body weight, preferably from about 50 μg to about 150 mg / kg body weight. Because each subject has varying degrees of manifestation and each agent has unique therapeutic properties, it is up to the expert to determine the subject's response to the treatment and to change the dosage accordingly.

The present invention also provides a food additive or beverage composition for the prevention or improvement of endoplasmic reticulum stress and / or mitochondrial function impairment containing a blank extract or imperitorin as an active ingredient.

When the blank extract or imperitorin of the present invention is used as a food additive, each of them may be added as it is or may be appropriately used according to conventional methods with other foods or food ingredients. The mixed amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment). In general, white paper extract or imperitorin may be added in amounts of 0.0001 to 30% by weight, preferably 0.1 to 10% by weight, based on the raw materials in the manufacture of foods or beverages, but the amount added may vary depending on the purpose. It can be adjusted. There is no particular limitation on the type of food. Examples of foods to which white paper extract or imperitorin can be added include meat, sausage, bread, chocolate, candy, snacks, confectionary, pizza, ramen, other noodles, dairy products including gums, ice cream, various soups, drinks, Tea, drink, alcoholic beverages and vitamin complexes.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited by the following examples.

Example

Low fat and high fat diet

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

White Paper Extract and Imperitorin Preparation

White paper extract was extracted by cold washing at room temperature with 70% ethanol, and the freeze-dried powder was used in water or buffer solution. Imperitorin, one of the main constituents of white paper, can be used by separating white paper with methanol or ethanol extract with chloroform, separating and identifying by using silica gel and column chromatography. Lean was used and the purity was 99.16%.

Induction of Vesicular Stress

To induce vesicle stress, Tunicamycin, which is known to induce vesicle stress by inhibiting N-glycosylation of newly synthesized protein in endoplasmic reticulum, or known to induce vesicle stress by destroying calcium homeostasis of 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. The optical density was measured at wavelengths of 260 nm and 280 nm, and the ratios were within the range of 1.8-1.95. RNA was quantified using the density value. 1 μgRNA was converted to cDNA using Maxime RT Premix Kit (Intron Biotechnology, Korea) and PCR using GeneAmp® PCR system 9700 (Applied Biosystems, USA) under optimal conditions using primers designed for each gene as shown below. Was performed. Each PCR product was subjected to electrophoresis on 1% agarose gel to measure relative concentration on UV using an image densitometer (Alpha Ease FC software (Alpha Innotech, USA)). MRNA levels were normalized to 18S rRNA levels. The primers used for RT-PCR are as follows.

Gene Primer sequence (from 5 'to 3') Product Length (bp) Anealing Temp. ( ^o C) GRP78

XBP1p

NPY

AgRP

18S rRNA
F-GAGATCATCGCCAACGATCAG
R-ACTTGATGTCCTGCTGCACAG
F-GGTCTGCTGAGTCCGCAGCAGG
R-GGGCTTGGTATATATGTGG
F-GCTTGAAGACCCTTCCATGTGGTG
R-GGCGGAGTCCAGCCTAGTGG
F-GAAGGCCTGACCAGGCTCTGTTCC
R-AAAGGCATTGAAGAAGCGGCAG
F-GAGCGAAAGCATTTGCCAAG
R-GGCATCGTTTATGGTCGGAA 188

335

142

251

101
55

60

60

60

60

Oxygen Respiration Rate (O ₂  consumption rate (OCR) measurement

Muscle tissue collected from the sacrificed mice was treated with 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 It was 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. Muscle tissue in BIOPS solution was treated with 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. Transfer to cold respiration buffer (pH 7.1) and increase cell membrane permeability with stirring for 10 minutes. After transferring muscle tissue with increased cell membrane permeability to Oxygraph-2k chamber maintained at 37 ^o C (Oroboros, Austria), the tissue was infused with 100 mM p1, p5-di (adenosine-5 ')-pentaphosphate into the chamber. Adenylation was prevented. OCR was measured by basal respiratory rate of each tissue and then measured oxygen consumption (OCR) by sequentially adding the substrate and inhibitor 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 OCR thus measured is expressed in pmoles / sec / mg wt.

Experimental Example 1 Effect of White Paper Extract or Imperitorin on Vesicular Stress

1) Effect of Hep G2 Cells on Apoptosis due to Vesicular Stress

Hep G2 cells in a solution containing 10% fetal bovine serum (FBS) in minimum essential medium (MEM) at 37 ° C, 5% carbon dioxide (CO ₂ ), 95% air (O ₂ ) Incubated with. Hep G2 cells (1 × 10 ⁴ cells / well) in culture were pretreated for 5 hours by adding white paper extract (ADEx, 0.5 μg / mL) or imperitorin (IMP, 0.1 μg / mL). Topicagagin (3 μg / mL) or tunicamycin (4 μg / mL), which induces endoplasmic reticulum stress, was added to each of the blank extract or imperitorin and non-treated groups, followed by incubation for 24 hours, followed by cell death recovery. The effect was measured by MTT assay. In cell viability compared to the control group (DMSO-treated group), which was not treated with both white paper extract and topsigin or tunicamycin, the white paper extract or imperitorin pretreatment group almost completely prevented the death of hepatocytes induced by endoplasmic reticulum stress. Was observed (FIG. 1).

2) Effect on vesicle stress marker gene expression in Hep G2 cells

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

3) Effect on Reduction of ATP Level by Antifoam Stress in Hep G2 Cells

Hep G2 cells in a solution containing 10% fetal bovine serum (FBS) in minimum essential medium (MEM) at 37 ° C, 5% carbon dioxide (CO ₂ ), 95% air (O ₂ ) Incubated with. Emperatorin (IMP; 0.1 μg / mL) was added to Hep G2 cells (1 × 10 ⁵ cells / well) in culture, followed by incubation for 5 hours, followed by Tophgagin (Thap; 3 μg / mL). ) Or tunicamycin (Tuni; 4 μg / mL) was added and incubated for 24 hours (Tab + IMP group or Tuni + IMP group in FIG. 4). However, no imperitorin was added to the control group (DMSO group, Thap group or Tuni group). ATP concentration was measured using an ATP-Bioluminescence assay kit (#FLASC; Sigma-Aldrich, USA) in all groups of cells (1 × 10 ⁵ cells / mL).

As shown in FIG. 4, in the IMP group added with only imperitorin, the ATP concentration showed a tendency to increase slightly compared to the control group, but there was no significant difference, and in the vesicle stress (Thap group, Tuni group), the ATP concentration was increased. It was found to decrease significantly. However, ATP concentrations were significantly increased when Thap and Tunicarmycin were treated with imperitorin together with Thap and Tuni groups. These results suggest that imperitorin increased ATP production by increasing mitochondrial activity by relieving both representative endoplasmic reticulum stresses.

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 diabetic model animals db / db mice were divided into two groups, the control group and the experimental group, respectively, and the control group was divided into four groups by receiving water (CTL) and the experimental group by extracting the blank paper (ADEx). Each group used four mice. All experimental animals received a 33-day free chaw diet. The experimental group was orally administered 100 mg / kg / day white paper extract once a day orally using oral-zonde, and the control group was administered the same amount of water in the same way.

1) weight change

Although the dietary intake of each group was not observed during the experimental period, the body weight of the lean mice showed almost 1.4 times higher body weight than the lean mice. db / db mice had a decrease in body weight gain after administration of white paper extract (FIG. 5).

2) Effect on epididymal fat

After the administration period of the white paper extract, the white fat portion of the testis was separated from the sacrificed mice and weighed. The administration of blank extract (ADEx) significantly decreased the amount of testis white fat in db / db mice, and it was also statistically insignificant in lean mice. (Figure 6)

3) Effect on Mitochondrial Activity

Mitochondria were isolated from the soleus muscle of sacrificed rats after the administration period of the white paper extract (Choi et al, 2005; Gogvadze et al., 2004), and the results of oxygen consumption (oxygen respiration rate) were measured. Shown in 7. Administration of white paper extract (ADEx) increased the mitochondrial activity of soleus muscle in mice in both lean mice (lean group) and db / db mice (db / db group). In particular, administration of imperitorin significantly increased the level of oxidative phosphorylation (OXPHOS) complexes in the case of disease (db / db group) than normal condition.

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 orally administered to measure blood glucose hourly (0, 15, 30, 60, 120 minutes) (Oral glucose tolerence test, OGTT oral glucose tolerance test) (Fig. 8).

In the db / db group, the blood glucose level was about 4-5 times higher than in the control group. However, the administration of blank extract (ADEx) increased insulin sensitivity by imperitorin, which resulted in a shorter decrease in blood glucose than the db / db group. It was. These results are the result of the administration of imperitorin for 33 days and may be better if long term imperitorin is administered. In addition, the short-term administration increased the initial insulin sensitivity, which may have a positive effect on the insulin secretion function of the pancreas.

Experimental Example 3 Effect of Imperitorin on High-Fat Diet Ingested Mice

Low-fat diet group (LF; # D12450B; Research diets, USA; 10% Kcal fat), high-fat diet group (HF; # D12492; Research) were 6-week-old male mice with C57BL / 6J (SLC, Japan). diets; 60% Kcal fat) and high-fat diet with 5 mg / kg / day or 10 mg / kg / day imperitorin (IMP), respectively (HF + IMP5 and HF + IMP10). Divided into groups. Each group used 7 mice. The experimental animals were fed low-fat diet and high-fat diet for 7 weeks, respectively. Imperatorin-treated groups received oral-zonde oral administration during the experimental period. After 7 weeks, the experimental animals bred as described above were sacrificed, and blood, brain, hypothalamus and liver, and soleus muscles were extracted and tested.

1) change in weight and dietary intake

Table 1 below shows the change in the average body weight of each experimental animal group, Table 2 and Figure 9 is the result of measuring the change in dietary intake rate.

Body weight and dietary intake rate (kcal / day) was increased in the experimental animals, and weight and dietary intake were decreased by administration of imperitorin (IMP).

2) Effect on Expression of Appetite-regulating Neuropeptides in Hypothalamus

In order to determine the cause of decreased dietary intake, the hypothalamus was isolated from each group of mice, and the expression of neuropeptide mRNA involved in appetite regulation was measured by RT-PCR. The appetite-stimulating neuropeptides NPN (neuropeptide Y) and AgRP (agouti-related protein) mRNAs were increased due to high-fat dietary intake, and their expression was reduced by imperitorin (IMP) administration (FIG. 10). . Therefore, imperitorin appears to have an appetite suppressing effect by inhibiting the expression of NPY and AgRP.

3) Effect on epididymal fat

High fat diets lead to weight gain and excess fat accumulation. White fat (epididymal fat) of the experimental animal group from testes After collection, the weight was measured. Excess fat accumulation following dietary intake of high fat significantly reduced white fat at high concentrations when administered with imperitorin (IMP) (FIG. 11). These results show that imperitorin is effective in reducing body fat accumulated by high fat diet.

4) Effects on fatty liver

Too much fat intake is more than enough to break down in the liver, so fat can no longer break down and fat builds up. Fatty liver becomes more severe, and liver fibrosis is progressed and eventually the liver cannot function properly. Therefore, when the high-fat diet was ingested by immunohistostaining (immunohistochemistry) to see the shape of the cells of the liver tissue is shown in Figures 12 and 13.

12 shows the results of confirming the expression of C / EBP-α, a protein that plays a major role in adipose metabolism and adipocyte differentiation. Overexpressed C / EBP-α due to high fat diet can be seen to normalize to similar levels as the control group when administered imperitorin.

In addition, Figure 13 is the result of confirming the shape of the hepatocytes through H & E staining. In the control group, the morphology of the cells remained intact, but in the high-fat diet group, the morphology of the cells was injured so much, and many lipid droplets were produced. When administration of imperitorin, dose-dependent normalization of hepatocytes similar to the control group was found.

These results show that impairitorin has an excellent effect on restoring liver function by inhibiting synthesis or accumulation of fat in the liver due to high fat diet.

5) Effect on Mitochondrial Activity

Mitochondrial oxygen respiration rate was measured using soleus muscle isolated from the test mice. Figure 14 shows that high fat diet did not significantly reduce the oxygen consumption of mitochondria, but administration of imperitorin improves the mitochondrial activity of muscle, especially the activity of OXPHOS complex I.

6) Oral glucose tolerance test (OGTT)

In order to measure insulin resistance, the experimental animals were kept on an empty stomach 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 is shown in FIG. 15. Fasting blood glucose was increased in the high-fat diet group compared with the normal diet group, and hyperglycemia persisted 2 hours after the administration of glucose, resulting in diabetes and insulin resistance. On the other hand, in the high-fat diet group administered imperitorin, fasting blood glucose was slightly decreased and blood glucose removal rate was increased after ingestion of glucose, but blood glucose was not normalized completely. These results show that the imperitorin does not completely normalize the increase in blood glucose and insulin resistance caused by a high fat diet, but may improve insulin resistance.

<Production Example>

It is possible to make products which can be administered through various routes, including the blank extract or imperitorin of the present invention, but are not limited thereto.

Capsule

White Paper Extract 20.0% (w / w) / Lactose 79.5% (w / w) / Magnesium Stearate 0.5% (w / w)

The ingredients are mixed and dispersed into capsules containing about 100 mg each.

refine

Imperitorin 20.0% (w / w) / Magnesium Stearate 0.5% (w / w)

Croscarmellose Sodium 2.0% (w / w) / Lactose 76.5% (w / w)

Polyvinylpyrrolidine (PVP) 1.0% (w / w)

The components are combined and granulated with a solvent such as methanol. Thereafter, the formulation is dried and tablets are formed with a suitable tablet press.

beverage

9% by weight of white paper extract, 10% by weight fructose, 1% by weight per white sugar, 0.2% by weight of sodium citrate was added to 100% by weight of purified water and stirred to prepare a conventional beverage production method.

references

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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.

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.

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.

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. Jaehun Kang, Namsun Kim. (2002) Obesity Trends in Korea. Korean Journal of Obesity , 11 (4), 329-336.

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

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

21. Kim Jong Kyun, Jung Soo Lim, Jun Jun, Sang Hyun Park, Doo Ho Hong. (2007). Differences in Adolescent Obesity in Incheon by Economic Level. Korean Journal of Obesity . 16 (2), 76-85.

22. Yoon Na Lee, Hae Shin Shin, Bok Hee Kim, Young Ae Jang, Cho Cho Il. (2004). Dietary Characteristics of Obese Population in Korea. Korean Society of Obesity Spring Conference

23. Hyemi Choi, Yeonkyung Seo. (2004). "Obesity and Metabolic Syndrome"-How is their diet? Korean Society of Obesity Spring Conference

      FIG. 1 shows the effect of blank extract (ADEx) or imperitorin (IMP) on apoptosis of Hep G2 cells by vesicle stress induced by Topsigagin or tunicamycin.

Figure 2 is a result showing the inhibitory effect of white paper extract (ADEx) on the expression of 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 imperitorin (IMP) on the expression of the vesicle stress marker genes GRP78 and XBP1p in Hep G2 cells.

FIG. 4 is a result showing the effect of imperitorin (IMP) on the reduction of ATP amount by endoplasmic reticulum stress (tapsigarin or tunicamycin) in Hep G2 cells.

Figure 5 is a result showing the effect on the weight change when administered the blank extract (ADEx) to diabetic mice.

Figure 6 is a result showing the effect on the testosterone white fat weight by administration of white paper extract (ADEx) to diabetic mice.

FIG. 7 shows the results obtained by measuring the amount of mitochondrial-activated oxygen in the soleus muscle of mice administered white paper extract (ADEx) using an Oxygraph-2k instrument.

8 shows the results of administration of white paper extract (ADEx) in oral glucose tolerance test of Lean mice and diabetic model (db / db) mice.

9 is a result of measuring the change in dietary intake by administration of imperitorin in high-fat diet and low-fat diet mice.

Figure 10 shows the effect of imperitorin on the expression of appetite promoting genes in the hypothalamus (hypothalamus) of the brain of the low-fat diet and high-fat diet mice.

Figure 11 shows the effect of imperitorin on the weight of white fat in low-fat diet and high-fat diet mice.

12 shows the results of immunohistochemistry confirming the effect of imperitorin on C / EBP-α expression in liver of low-fat diet and high-fat diet mice.

FIG. 13 shows the results of confirming the morphology of cells according to the administration of imperitorin using H & E staining in the liver of low-fat diet and high-fat diet mice.

FIG. 14 shows the results of measuring mitochondrial oxygen consumption according to whether administration of imperitorin in soleus muscle collected from low-fat diet and high-fat diet mice using an Oxygraph-2k device (Oroboros, Austria).

15 is a result of measuring the oral glucose tolerance according to whether administration of imperitorin in low-fat diet group and high-fat diet group mice.

Claims (7)

Obesity, insulin resistance, body fat increase, fatty liver, liver fibrosis, metabolic syndrome, lipid metabolism, hypertension, neurodegenerative disorders, bipolar disorder, diabetes, arteriosclerosis, ischemia, including white paper extract and pharmaceutically acceptable carrier, Pharmaceutical composition for the prevention or treatment of heart disease, liver disease, pancreatic disease, cancer, aging, cardiovascular disease, Parkinson's disease, Huntington's disease, dementia or spinal cord injury. Obesity, insulin resistance, body fat gain, fatty liver, liver fibrosis, metabolic syndrome, lipid metabolism, hypertension, neurodegenerative disorders, bipolar disorder, including imperitorin or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier Pharmaceutical composition for preventing or treating diabetes, arteriosclerosis, ischemia, heart disease, liver disease, pancreatic disease, cancer, aging, cardiovascular disease, Parkinson's disease, Huntington's disease, dementia or spinal cord injury. 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). Food composition for reducing vesicle stress and / or improve mitochondrial function comprising a blank extract or imperitorin as an active ingredient. Containing blank extract or imperitorin as an active ingredient, vesicle stress relief and / or beverage composition for improving mitochondrial function. Obesity, insulin resistance, body fat increase, fatty liver, liver fibrosis, metabolic syndrome, lipid metabolism, hypertension, neurodegenerative disorders, bipolar disorder, diabetes, arteriosclerosis, ischemia Food composition for preventing or improving heart disease, liver disease, pancreatic disease, cancer, aging, cardiovascular disease, Parkinson's disease, Huntington's disease, dementia or spinal cord injury.

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