KR101253029B1 - Activator for AMPK - Google Patents

Abstract

The present invention is an AMPK (5 'adenosine monophosphate-activated protein kinase) activator containing the extract as an active ingredient and one or more symptoms selected from obesity, diabetes, metabolic syndrome, degenerative disease or cancer containing the extract as an active ingredient Or a composition for treating, ameliorating or preventing a disease. The activator of the present invention is effective for AMPK activation, and the composition of the present invention is effective for treating, ameliorating or preventing one or more symptoms or diseases selected from obesity, diabetes, metabolic syndrome, degenerative disease or cancer.

Description

AMPM Activator {Activator for AMPK}

The present invention relates to an AMPK activator, and more particularly, to an AMPK activator effective for the treatment, improvement or prevention of obesity, diabetes, metabolic syndrome, degenerative disease and cancer.

AMPK activators are substances that increase the activity of AMPK (5'AMP-activated protein kinase; 5 'adenosine monophosphate-activated protein kinase), and AMPK is an enzyme that plays an important role in cellular energy homeostasis. By activating AMPK, it is known that it can have a prophylactic or therapeutic effect on diabetes, metabolic syndrome, obesity, cancer, degenerative diseases (dementia), etc., and research on AMPK activators is ongoing. Implications for human health and disease. Biochem J 418 (2), 261-275 (2009), Mechanisms linking obesity, chronic kidney disease, and fatty liver disease: the roles of fetuin-A, adiponectin, and AMPK. J Am Soc Nephrol 2010 Mar .; (21) 3: 406-12}.

Thus, the development of AMPK activators is needed.

One technical problem to be solved by the present invention is to provide an AMPK activator.

Another technical problem to be solved by the present invention is to provide a composition for the treatment, improvement or prevention of one or more symptoms or diseases selected from obesity, diabetes, metabolic syndrome, degenerative disease or cancer.

The present invention provides an AMPK (5 'adenosine monophosphate-activated protein kinase; 5' adenosine monophosphate-activated protein kinase) activator containing the extract (蒼朮; Atractylodis Rhizoma) as an active ingredient.

The creation may be dried root of the perennial herb, Atracthlodes japonica KOIDZ.

The creation extract may be one or more solvent extracts selected from alcohols such as methanol or ethanol or water, preferably water extracts, may be extracted or purchased by a known method such as hot water extraction.

The creation extract may comprise 1 to 10% by weight relative to the total weight of the activator, there is a fear that the effect is not sufficient at less than 1% by weight, there is a possibility that the effect is no longer greater than 10% by weight.

The AMPK activator of the present invention is not limited thereto, but may specifically be for treating, ameliorating or preventing one or more selected from obesity, diabetes, metabolic syndrome, degenerative disease, or cancer. The diabetes may be type 2 diabetes, and the degenerative disease may be dementia, preferably Alzheimer's dementia {AMPK and the biochemistry of exercise: Implications for human health and disease. Biochem J 418 (2), 261-275 (2009), Mechanisms linking obesity, chronic kidney disease, and fatty liver disease: the roles of fetuin-A, adiponectin, and AMPK. J Am Soc Nephrol 2010 Mar .; (21) 3: 406-12}.

The present invention also provides a composition for the treatment, improvement or prevention of one or more symptoms or diseases selected from obesity, diabetes, metabolic syndrome, degenerative disease, or cancer containing the extract as an active ingredient. The creation extract may be one or more solvent extracts selected from alcohols such as methanol or ethanol or water, preferably water extracts, may be extracted or purchased by a known method such as hot water extraction. The diabetes may be type 2 diabetes and the degenerative disease may be dementia, preferably Alzheimer's dementia.

The creation extract may comprise 1 to 10% by weight relative to the total weight of the composition, there is a fear that the effect is not sufficient at less than 1% by weight, there is a possibility that the effect is no longer greater than 10% by weight.

The treatment, improvement or prevention may be by AMPK activation.

The AMPK activator or composition of the present invention may be a pharmaceutical composition or a food composition. The food composition may be a health functional food, the formulation is prepared according to a conventional method, and then dried with a carrier and then encapsulated or may be formulated in the form of other tablets, granules, powders, beverages, porridge, and the like. In addition to the above, it may be in any food form. The pharmaceutical composition may be for the treatment or prevention of one or more symptoms or diseases selected from obesity, diabetes, metabolic syndrome, degenerative disease, or cancer, wherein the food composition is one selected from obesity, diabetes, metabolic syndrome, degenerative disease, or cancer It may be for improving or preventing the above symptoms or diseases.

The creation extract exhibits an AMPK activating effect and / or treatment, amelioration or prophylactic effect of one or more symptoms or diseases selected from obesity, diabetes, metabolic syndrome, degenerative disease, or cancer.

Accordingly, the present invention provides a AMPK activating use of a production extract and / or treatment, amelioration or prevention of one or more symptoms or diseases selected from obesity, diabetes, metabolic syndrome, degenerative disease, or cancer.

In addition, the present invention provides an AMPK activator of the production extract and / or the use of the production extract for the treatment, amelioration or prevention of one or more symptoms or diseases selected from obesity, diabetes, metabolic syndrome, degenerative disease, or cancer.

In addition, the present invention provides a method for AMPK activation and / or obesity, diabetes, metabolic syndrome, degenerative disease, or cancer, comprising administering to a mammal (including human) in need of a pharmaceutically effective amount of the extract. Provided are methods for treating, ameliorating or preventing one or more selected symptoms or diseases.

Unless stated otherwise, the contents of the AMPK activators of the present invention, the compositions, uses, and methods of the present invention apply equally unless they contradict each other.

The creation extract and the AMPK activator and / or composition comprising the same can be administered orally or parenterally to mammals, including humans, it can be administered in combination with an pharmaceutically acceptable carrier to formulate and administer. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants which are commonly used may be used. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid form preparations may include at least one excipient such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. It is prepared by adding. Lubricants such as magnesium and talc are also used. Liquid preparations for oral administration include suspensions, solvents, emulsions and syrups, and may include various excipients such as wetting agents, sweeteners, fragrances and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. have. Formulations for parenteral administration include injectable solutions, suspensions, emulsions, lyophilizers, nasal washes and suppositories. Injectable solutions, suspensions and emulsions can be prepared by mixing water, non-aqueous solvents or suspending solvents with the active ingredient. As non-aqueous solvents and suspending solvents, vegetable oils such as propylene glycol, polyethylene glycol and olive oil, and ethyl oleate Injectable esters such as and the like.

As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol, gelatin and the like can be used. The generating extract, AMPK activator or composition comprising the same, can be administered by subcutaneous injection, intravenous injection or intramuscular injection during parenteral administration.

In the AMPK activator, composition, use and method of the present invention, it can be used at a dose of 150 ~ 300 mg / kg per adult basis on the basis of the extract extract.

The extract may be formulated by adding a pharmaceutically or food-acceptable carrier, excipient or diluent, etc. For formulation, see Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA, et al. Reference may be made.

AMPK activator of the present invention is excellent in AMPK activating effect, the composition of the present invention is effective in the prevention or treatment of obesity, diabetes, metabolic syndrome, degenerative diseases, cancer and the like.

1 is a diagram showing the results of confirming the expression of the activity of AMPK and ACC by Western blot method.
Figure 2 is a diagram showing the results of the RT-PCR experiment.
Figure 3 is a graph (B) showing the weight change for the experimental animals of Example 1-2 and the photo (A) before and after opening to confirm the amount of visceral fat of the experimental animal at the end of the experiment 1-2. .
Figure 4 is a graph (B) showing the fat cell photograph (A) and the distribution of fat cells by size as a result of the <fat shape observation> experiment of Example 1-2.

Hereinafter, the present invention will be described in more detail with reference to Examples and Preparation Examples, but the following Examples and Preparation Examples are only for illustrating the present invention, and the content of the present invention is not limited to the following Examples or Preparation Examples.

In the following examples, the statistical processing of the data was performed using a one-way analysis of variance at p < 0.05, p <0.01, and p < 0.001 with mean ± standard deviation calculated using the SPSS 14.0 statistical program. The significance between each group was tested by LSD (least significant difference test).

<Example 1> AMPK activation effect (anti-obesity effect) confirmation of the extract

1-1. Preparation of Generating Extracts

After extracting with 100 liters of hot water (100 degrees Celsius) per 100 g of water (Atractylodis Rhizoma, Musk, Gyeongdong Market, Seoul, South Korea), the filtrate was concentrated under reduced pressure (60 degrees Celsius), and then freeze-dried ( Freeze-dried with EYELA, FDV-1200) to prepare the resulting extract. In the experiment, the extract was used by dissolving in tertiary distilled water (DDW).

1-2. Preparation and Processing of Animals

<Preparation and Administration of Animals>

Five-week-old male ICR (Imprinting Control Region) mice (Orient, Seongnam, Korea) were stored in a plastic mouse cage and stored in a laboratory at 23 ± 2 ℃, relative humidity 50 ± 10%, light / dark cycle (12/12) hr) was bred under the conditions, and water and feed were freely ingested for 1 week before the experiment was used for the experiment. The dietary methods of overfeeding and energy dense diets were provided and transformed into a diet-induced obese (DIO) model that induced obesity. The experimental group included the regular diet (10% Kcal% fat, Research DIETS) and the high diet (45% Kcal% fat, Research DIETS) diet and high fat diet (45% Kcal% fat, Research DIETS). Fat Diet (weakly referred to as HFD), high fat diet and the production group of Example 1-1 (150 mg / kg) administered with the extract (150 mg / kg) of Example 1-1 (weakly referred to as HFD + AR150 or AR150) ), And divided into high fat diet and Example 1-1 (300 mg / kg) administration group (weakly referred to as HFD + AR300 or AR300) administered with the extract (300 mg / kg) of Example 1-1. Each experimental group was divided into six animals, and the extract of Example 1-1 was administered orally daily for 5 weeks with a high fat diet, and body weight and blood glucose were measured twice a week. Body weight was measured with a scale (OHAUS, Adeventurer Pro AVG812), blood glucose was measured with a SMARTLAB (Erba, USA) instrument.

Blood Index Analysis

Blood collection for blood index analysis was performed after 12 days of fasting and 12 hours of fasting. The collected blood was centrifuged at 5,000 rpm for 5 minutes and serum was used for analysis. Blood glucose, triglycerides, nonesterified fatty acid (NEFA), and serum index indicator sALT (serum aspartate aminotransferase) were purchased as measurement items (Stanbio, USA), and the biochemical analyzer (SMARTLAB, USA) Measured according to the instructions. Blood insulin concentration was measured by purchasing a mouse insulin ELISA kit (Shibayagi, Japan) and using an ELISA reader (Labsystems, Finland) according to the manufacturer's instructions. In addition, homeostasis model assessment of insulin resistance (HOMA-IR), an insulin resistance index, was calculated. The calculation method is Insulin (μU / mL) × glucose (mM) /22.5.

<Local form observation>

At the end of the experiment, epididymal adipose tissue collected from experimental animals was fixed using 10% neutral buffered formalin, and paraffin blocks were prepared by dehydration and embedding, and 5 µm thick tubular sections were prepared. Subsequently, paraffin is removed using xylene, hydrophilized with 100%, 95%, 90%, 80%, and 70% alcohol, stained with hematoxylin and eosin, and dehydrated and sealed with Canada balsam. And optical microscope (Olympus, Japan).

In addition, the number of fat cells by size was counted by the IMAGE-PROPLUS program.

<RNA Extraction and RT-PCR>

At the end of the experiment, total RNA was isolated from epididymal adipose tissue collected from experimental animals using guanidine thiocyanate-water saturated phenol / chloroform. Total RNA in the water layer was precipitated using isopropyl alcohol, and the isolated RNA was quantified by measuring absorbance at wavelengths of 260 nm and 280 nm, and 1 μg of total RNA was obtained using Moloney murine leukemia virus transcriptase and Oligo (dT) 15 primer. Reverse transcription. Primer types and sequences are as shown in Table 1 below. Primer was added to 25 μL of the reaction solution containing 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl 2, 0.5 mM dNTP, 5 μL cDNA and 2.5 units of Taq DNA polymerase so that the final concentration was 0.5 μM. It was. PCR conditions were denatured for 1 minute at 95 degrees Celsius, paste for 30 seconds (corresponding paste temperature is shown in Table 1), and extended for 1 minute at 72 degrees Celsius for a total of 30 cycles. The reaction product was then electrophoresed at 100 V using a 1% agarose gel stained with 0.5 μg / ml ethidium bromide. Beta-actin was used as a control for the amplified genes.

[Table 1]

Protein Extraction and Western Blot

At the end of the experiment, the epididymal adipose tissue from the animal was homogenized with lysis buffer (50mM Tris-HCl pH 7.5, 1mM EDTA, 0.25% sucrose, 0.4mg / ml digonin, 1.5mM PMSF) and the protein was then centigrade. Centrifugation was carried out at 12000 rpm for 20 minutes at 4 degrees. Protein quantitation was measured by Bio-Rad assay reagent (Bio-Rad, USA), 30μg of the analyzed protein was separated by SDS-PAGE, transferred to gel membrane and blocked for 1 hour at room temperature with 5% skim milk. (blocking) and then diluted with a primary antibody (P-AMPK, Cell Signaling, 2531; AMPK, Cell Signaling, 2532; P-ACC, Cell Signaling, 3661; ACC, Cell Signaling, 3662) It was reacted overnight at 4 degrees. Next, the cells were washed three times with Tris-buffered saline tween-20 (TBST) and then reacted with a secondary antibody (Santa Cruz, sc-2313) diluted 1: 2000 at room temperature for 1 hour. It was then washed three times with TBST and developed on an X-ray film using ECL solution (Amersham, Sweden).

1-3. AMPK activation effect confirmed

<Protein extraction and Western blot> experiments of 1-2. Are shown in Figure 1. 1 is a diagram showing the results of confirming the expression of the activity of AMPK and ACC by Western blot method. 1, pAMPK refers to the amount of phosphorylated AMPK protein, AMPK refers to the base level of AMPK protein. Similarly, pACC means the amount of phosphorylated ACC protein, ACC means the amount of ACC protein at base level, and Actin means control protein as loading control. As shown in FIG. 1, pAMPK and pACC proteins were hardly expressed in the RD and HFD groups, but dose-dependently increased in the production extract administration group (AR150 and AR300).

From the results it can be seen that the extract extract increases the phosphorylation of AMPK in a dose-dependent manner. The resulting extract can be seen to activate AMPK. Acetyl-CoA carboxylase (ACC), a substrate of AMPK, is a major enzyme in fatty acid synthesis and its activity is inhibited when phosphorylated by AMPK. ACC is a rate-limiting enzyme that regulates malonyl-CoA synthesis, which plays an important role in the inhibition of fatty acid oxidation in mitochondria. AMPK activation inhibits ACC and consequently reduces malonyl-CoA, thereby promoting fatty acid oxidation.

Therefore, it can be seen that the creation extract promotes AMPK phosphorylation in adipocytes, resulting in fatty acid oxidation, thereby reducing the size of adipocytes, thereby exhibiting an anti-obesity effect.

In addition, by activating the AMPK by the production extract, by inhibiting the key enzymes such as ACC in fatty acid synthesis, further use of ATP is suppressed and related diabetes, such as obesity, metabolic syndrome, type 2 diabetes, dementia It can be seen that it is effective in the treatment, improvement or prevention of degenerative diseases such as Alzheimer's dementia, and symptoms or diseases such as cancer.

1-4. Confirmation of adifferentiation-related gene expression inhibitory effect

<RNA extraction and RT-PCR> experiment results of 1-2 are shown in FIG. 2. Many genes are known to be expressed when adipocytes are differentiated, and representative transcription factors include peroxisome proliferator-activated receptor γ (PPAR-γ) and CCAAT / enhancer binding protein α (C / EBPα). Genes such as fatty acid binding protein (aP2), lipoprotein lipase (LPL), fatty acid syntase (FAS) and stearoyl-CoA desaturase-1 (SCD-1) are known. Inhibition of the expression of these genes indicates that adipocyte differentiation is inhibited, resulting in an anti-obesity effect.

Figure 2 is the result of measuring the amount of gene expression related to differentiation. As shown in FIG. 2, the HFD group significantly increased the expression of adipose differentiation-related genes compared to the RD group, while in the AR150 and AR300 administration groups, the expression of genes was dose-dependently suppressed compared to the HFD group. Therefore, it can be seen that the creation extract inhibits the expression of adipose differentiation-related genes, and thus has an effect such as anti-obesity.

1-5. Check the effect of suppressing visceral fat and weight gain

Experimental animals of 1-2. Was opened at the end of the experiment was confirmed the amount of visceral fat, and photographed the results are shown in Figure 3 (A) with a photo before opening. In addition, the weight change for the experimental animals of 1-2. Is shown in Figure 3 (B). In addition, the following table shows the weight and the increase in weight after the start and end of the experiment.

[Table 2]

As shown in Figure 3 (A) high-fat diet group (HFD) showed the most visceral fat when opened compared to the normal diet group (RD). On the other hand, the production extract-treated mice were thinner and less visceral fat than the high-fat diet group. In addition, as shown in FIG. 3 (B), the weight of the high-fat diet group was not significantly different between the experimental groups before the induction of the high-fat diet, but the weight of the high-fat diet group began to increase significantly after 10 days of feeding. As time went by, it got bigger. In addition, as shown in the table, the high-fat diet group gained 263% more weight than the normal diet group, whereas the creation extract administration group (AR150, AR300) was 35.2% (P <0.01) and 49.2% (P <0.001), respectively, compared to the high-fat diet group. ) Weight gain was suppressed. That is, it can be seen that the weight decreases depending on the dose of the creation extract.

Therefore, it can be seen that the creation extract has an anti-obesity effect, such as preventing obesity induced by high fat diet.

1-6. Check effects such as anti-obesity

<Fat morphology observation> experimental results of 1-2 are shown in FIG. Figure 4 (A) is a photograph of the adipocytes, Figure 4 (B) is a graph showing the distribution of fat cells by size. As shown in FIG. 4 (A), the size of adipocytes was much larger in the HFD group than in the RD group, while the adipocytes were dose-dependently smaller in the AR150 and AR300 administration groups than in the HFD group, and the number of adipocytes by area was compared. As can be seen from FIG. 4 (B), the number of adipocytes having a larger area was larger in the HFD group than the RD group, but the number of adipocytes having a larger area than the HFD group was decreased in the AR150 and AR300 administration groups. From this, the extract was found to reduce the size of the white fat epididymal fat has the effect of preventing weight gain.

1-7. Confirm prevention effect such as diabetes

Blood glucose analysis, blood glucose, blood insulin concentration, and HOMA-IR in the experimental results of 1-2. Are shown in Table 3 below.

[Table 3]

In the blood glucose group, the HFD group increased 255% compared to the RD group, whereas the AR150 and AR300 groups significantly decreased blood sugar levels by 13.6% and 38.4%, respectively. Insulin levels also increased by 209% in the HFD group compared to the RD group, whereas insulin levels decreased by 11.1% and 33.5% in the AR150 and AR300 groups compared to the HFD group, respectively. The high-fat diet group showed a 5.4-fold increase in insulin resistance index (HOMA-IR) compared to the normal diet group, while the extract extract group showed a dose-dependent effect of 22.3% and 62.8% reduction, respectively. This result shows that insulin resistance, which is rapidly increased by high-fat diet, is very effectively blocked in the production extract group.

Therefore, it can be seen that the extracts are effective in the treatment or prevention of diabetes such as type 2 diabetes, metabolic syndrome, and degenerative diseases such as dementia, preferably Alzheimer's dementia, by reducing blood sugar, decreasing blood insulin concentration, and improving insulin sensitivity. .

1-8. Confirmation of inhibitory effect such as blood lipid increase

The blood index analysis of 1-2. NEFA, triglyceride (TG), the enzyme sALT measurement results of liver function are shown in Table 4 below.

[Table 4]

In the case of serum NEFA concentration, the HFD group increased 204% compared to the RD group, while the AR150 and AR300 administration groups showed 60.6% and 60.5% decreases, respectively. In triglycerides, the HFD group showed a significant increase of 233% compared to the RD group, while the AR150 and AR300 groups showed a 38.7% and 44.6% decrease, respectively, compared to the HFD group. SALT, an indicator of liver function, was significantly increased in the high-fat diet group compared with the normal diet group, and significantly decreased by 64.7% and 67.5% in the AR150 and AR300-administered groups, respectively.

Therefore, the extract extract shows the activation of AMPK, a protein corresponding to the metabolic sensor, and the resulting fatty acid oxidation, blood glucose and lipid profile, and the like, and associated with obesity, diabetes mellitus, type 2 diabetes, metabolic syndrome, and dementia. Preferably, it can be seen that it is effective in treating or preventing degenerative diseases such as Alzheimer's dementia and cancer.

Preparation Example 1 Preparation of Pharmaceutical Composition

Preparation of Example 1-1 A capsule was prepared by filling 300 mg of extract, 100 mg of corn starch, 100 mg of lactose, and 2 mg of magnesium stearate into a gelatin capsule.

Preparation Example 2 Preparation of Food Composition

Example 1-1 Add water (4 wt%), liquid fructose (0.5 wt%), oligosaccharide (2 wt%), sugar (2 wt%), and salt (0.5 wt%) to add the remaining amount After adjusting, homogeneously blended and sterilized to prepare a health drink.

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Claims (7)

Food composition for improving or preventing diabetes containing the extract as an active ingredient. The food composition of claim 1, wherein the extract is a water extract. delete delete A pharmaceutical composition for treating or preventing diabetes, comprising the extract as an active ingredient. The pharmaceutical composition for treating or preventing diabetes of claim 5, wherein the extract is a water extract. delete

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