KR102095199B1 - Method for activation of AMP dependent protein kinase using avenanthramide C - Google Patents

Abstract

The present invention relates to a method for activating AMPK, and more particularly, to a method for activating AMPK using Abenanthramide C and its derivatives extracted from oats.
According to the present invention, avenanthramide C (Avn-C) or a derivative thereof extracted from oats can activate AMPK (AMP-activated protein kinase) by binding to adrenaline receptor-α1a (AR-α1a).

Description

{Method for activation of AMP dependent protein kinase using avenanthramide C}

The present invention relates to a method for activating AMPK, and more particularly, to a method for activating AMPK using Abenanthramide C and its derivatives extracted from oats.

Recently, metabolic disorders due to complex causes such as obesity, diabetes, hyperlipidemia and cardiovascular diseases have increased exponentially. Among them, the incidence of metabolic diseases due to the imbalance of energy homeostasis is the highest. It is known that the abnormality of energy metabolic regulation is due to insulin resistance, but a detailed regulation mechanism for this is unknown.

According to a recent study, AMPK (AMP-activated protein kinase) centers on energy recognition and homeostasis control in vivo, and has been reported to play an important role in the metabolism of sugar and fat. The abnormality of the energy sensor has been shown to be highly associated with metabolic diseases, cardiovascular diseases, and cancer.

AMP activation protein kinase (AMPK) is an enzyme activated by adenosine monophosphate (AMP), and is an enzyme that acts as a sensor for maintaining energy homeostasis in cells. AMPK is activated when metabolic stress or exercise decreases energy in cells, that is, when ATP is depleted and the AMP / ATP ratio increases, inhibiting the process of consuming ATP and producing ATP (for example, Fatty acid oxidation and glycolysis).

The effect on the activation of AMPK is involved in target organs (liver, muscle, fat, pancreas, etc.), which are closely related to energy metabolism regulation. For example, when AMPK is activated in the liver, it inhibits the synthesis of fatty acids and cholesterol and promotes the oxidation of fatty acids. When AMPK is activated in skeletal muscle, it promotes oxidation of fatty acids and sugar absorption, and inhibits fat breakdown and fat production in fat cells. In addition, activation of AMPK in pancreatic β cells promotes insulin secretion. In relation to the role of AMPK, many studies have been conducted as drug targets for metabolic diseases.

As such, there are also patents for treating metabolic diseases by activating AMPK. Korean Registered Patent No. 1062670 discloses a composition for preventing or treating obesity-related diseases mediated by activation of AMPK containing 2,5-bis-aryl-3,4-dimethyltetrahydrofuran lignan as an active ingredient, , Korean Patent Registration No. 1063895 discloses that a peptide having an amino acid sequence containing Pro-Gly has anti-obesity and anti-diabetic effects by promoting energy metabolism of AMPK activity of skeletal muscle cells. In addition, Korean Patent Registration No. 1399313 discloses an effect of improving metabolism by culturing cells in a microgravity state in which there is little gravity to activate intracellular AMP activation protein kinase.

Thus, the present inventors confirmed that it is possible to activate AMPK when using oat extract avenanthramide C as a result of earnest research efforts to activate AMPK, a drug target of metabolic disease, and completed the present invention.

KR 10-1062670 B KR 10-1063895 B KR 10-1399313 B

Accordingly, it is an object of the present invention to provide a method for preventing or treating AMPK associated with activation of AMPK by activating AMPK using oat extract avenanthramide C.

The present invention to solve the above problems,

It provides a method of activating AMP-activated protein kinase (AMPK) by using Abenanthramide C (Avn-C) extracted from oats or a derivative thereof.

AMPK is an enzyme that acts as a sensor for maintaining energy homeostasis in cells. Activation of AMPK is involved in target metabolism, which is closely related to the regulation of energy metabolism. For this reason, drug research of metabolic diseases targeting activation of AMPK has been actively conducted, and as a result of the study by the present inventors to activate AMPK, Avenanthramide C (Avn-C) extracted from oats or a derivative thereof Found to activate AMPK, and completed the present invention.

As used herein, the term "oat" is estimated to have spread from eastern to central Europe with barley around 5000 BC, and is a plant that has been used in Europe for more than 800 years for nutritional and medical purposes. Oat is a monocotyledonous plant of the rice family, a perennial edible cultivation crop, also known as lotus, there are about 70 species, but only a few of them are cultivated, and mainly Wild oat ( avena sativa L.). The height is about 90cm, the leaves are 15 ~ 30cm long, the butterflies are 6 ~ 12㎜ long, flat, long vines are short, and the stalk is short and finely divided. Flowers bloom in May-June, and are cone-shaped inflorescences, branches turn and split again. The foreskin has no ridges, has many veins, and opens to both sides. Youngwa is wrapped in inner and outer spirits, has hairs, and has grooves on one side. Fruits are made from oatmeal and used as raw materials for alcohol, sweets, and feed for livestock.

The ingredients of the oats include beta-carotene, vitamin C, niacin, riboflavin, thiamine, retinol, dietary fiber, avenanthramide, and the like, among which avenanthramide inhibits free radicals as an antioxidant. It is said to be. The avenanthramide may be classified into avenanthramide-A, avenanthramide-B, avenanthramide C, avenanthramide-O, and avenanthramide-P.

The avenanthramide C of the present invention uses oat, a conventional plant extraction method known in the art, such as hot water extraction, organic solvent extraction, cold saliva extraction, ultrasonic extraction, reflux cooling extraction, and the like. The oat extract extracted through the organic solvent extraction method may be extracted using silica gel column chromatography and high performance liquid chromatography, but is not limited thereto. In the present invention, Abenanthramide C was purchased and used for this experiment.

In the method for activating AMPK of the present invention, AMPK is characterized in that Avn-C or a derivative thereof is activated by binding to adrenaline receptor-α1a (AR-α1a).

In the method of activating the AMPK of the present invention, the AMPK activated by binding Avn-C or a derivative thereof to the adrenaline receptor-α1a (AR-α1a) increases the level of p-GSK3β (Ser9) level, and p-IKK , cleaved caspase-3, and p-p65.

AMPK activity in the brain is primarily regulated by the α-adrenergic receptor (α-AR). In ligand binding, α-ARs are activated by Liver Kinase B1 (LKB1), calcium / calmodulin-dependent kinase or increased AMP levels, and then induce phosphorylation reaction and activity of AMPK. It was confirmed that the catecholamine ligand of α-AR (epinephrine or norepinephrine) shares structural similarity with Avn-C, particularly a portion of catechol. Therefore, the present inventors hypothesized that AMPK will be activated by Avn-C binding to α-AR because the catecholamine ligand of α-AR and Avn-C are structurally shared, and through this, a mechanism for activation of AMPK I tried to find out.

According to an embodiment of the present invention, as a result of using bead fractions immunoblotting to confirm that Avn-C binds to α-AR, it was confirmed that Avn-C binds only to α-1A among adrenaline receptors (Example 2) And Figure 3).

In addition, in order to confirm whether AMPK is activated by the above-described binding, the active foams of IKK, NF-κB-p65, GSK3β (Ser9), and caspase-3, which are factors in which the active foam is changed by activation of AMPK, are immunoblotting. As a result of analyzing the concentration and analysis, it was confirmed that the active form of the factors was changed when the phenoxybenzamine was not treated while the active form of the factors was not changed when the α-AR inhibitor phenoxybenzamine was treated. (See Example 3 and Figures 4 and 5). These results show that Avn-C activates AMPK by binding to α-1A.

In the method for activating AMPK of the present invention, Avn-C or a derivative thereof is activated by binding to adrenaline receptor-α1a (AR-α1a), and is characterized by inhibiting inflammatory factors.

In the method for activating AMPK of the present invention, the inflammatory factor is interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and cyclo-oxygenase-2 (COX-2). Is done.

According to the embodiment of the present invention, the level of phosphorylated AMPK was decreased in the case of hippocampal fragments treated with vehicle as a control, but it was confirmed that the level of phosphorylated AMPK was increased in the case of hippocampal fragments treated with Avn-C (implementation) See Example 1 and Figures 1 and 2). These results show that Avn-C can activate AMPK by binding to the adrenaline receptor-α1a, and can also suppress inflammatory factors as AMPK is activated.

As described above, according to the present invention, avenanthramide C (Avn-C) or a derivative thereof extracted from oats activates AMP-activated protein kinase (AMPK) by binding to adrenaline receptor-α1a (AR-α1a). You can.

1 shows the results of immunoblotting according to Example 1.
2 shows the results of immunofluorescence according to Example 1.
Figure 3 shows the results of Western blotting according to Example 2.
4 and 5 show the results of Western blotting according to Example 3 (FIG. 4; hippocampus of Tg2576 mice, FIG. 5; hippocampus of 5XFAD mice).

Hereinafter, the present invention will be described in more detail through examples. Since these examples are only for illustrating the present invention, it should not be construed that the scope of the present invention is limited by these examples.

<Experiment Animal>

C57Bl / 6J mouse, Tg2576 (APP KM670 / 671NL, 7-8 months old, Taconic) mouse, and 5XFAD (APP KM670 / 671NL [Swedish], APP I716V [Florida], APP V717I [London], PSEN1 M146L, PSEN1 L286V , 3-4 months old, Jackson Laboratory) mice were used, and the diet and drinking water were freely consumed in a controlled breeding room with a temperature of 20-30 ° C and a light / dark cycle of 12 hours. Used for.

Management and maintenance of laboratory animals and experimental procedures were conducted under the approval of the Chonnam National University Animal Experimental Ethics Committee.

Avenanthramide C (Avn-C; Avenanthramide-C) used in the present invention was purchased from Sigma-Aldrich and used in experiments.

<Hippocampal section of the example and Abenanthramide>

After anesthesia, the mouse was excised from the brain after decapitation. The extracted brain is transferred to cooled cerebrospinal fluid (aCSF; 124 mM NaCl, 3 mM KCl, 26 mM NaHCO ₃ , 1.25 mM NaH ₂ PO ₄ , 2 mM CaCl ₂ , 1 mM MgSO ₄ , and 10 mM glucose).

A hippocampus section was re-established in the brain to prepare a section with a thickness of 400 μm, and aCSF (124 mM NaCl, 3 mM KCl, 26 mM NaHCO ₃ , 2 mM CaCl ₂₎ was supplied while supplying a gas mixed with 95% oxygen and 5% carbon dioxide at room temperature. , 1mM MgSO ₄ , 10mM Glucose, 1.25mM NaH ₂ PO ₄ , and 2mM CaCl ₂ ) were stabilized for 1 hour and used in the experiment.

Example 1. Confirmation of AMPK activation effect of Avn-C

The AMPK activation effect of Avn-C was confirmed by immunoblotting and immunofluorescence.

The hippocampal tissue lysate used for immunoblotting is RIPA buffered in a hippocampal section treated with Vehicle and Avenanthramide C, crushed using a homogenizer on ice, and then RIPA [50 mM Tris- for 30 minutes at 4 ° C. It was prepared by dissolving with HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM PMSF (phenylmethylsulfonyl fluoride), 1 mM DTT, and 2 μg of leupeptin and aprotinin, respectively.

The crushed cells were centrifuged at 12,000 g at 4 ° C. for 20 minutes, and the resulting soluble fraction was denatured in Laemmli buffer and SDS-PAGE was performed. Protein concentration is confirmed using BCA analysis. Proteins were separated on 10% -12% gels and transferred to PVDF membranes (Millipore, Bedford, MA, USA). The membrane was blocked with amresco, a rapid block solution solution (pH 7), and blotting was performed using enhanced chemiluminescence (ECL). Antibodies to AMPK and antibodies to phosphorylated AMPK were used for blotting.

In addition, immunofluorescence (IF) was performed by cutting mouse brain tissue embedded in paraffin to a thickness of 4 um, followed by deparaffinization. First, it was boiled at 125 ° C in 10 mM pH 6.0 Tris buffer, cooled sufficiently, and treated with 3% H ₂ O ₂ for 30 minutes to suppress the activity of endogenous peroxidase. After sufficient washing, after treatment with a protein blocking agent (Millipor) for 30 minutes to suppress cross-reactivity of heterologous proteins, NeuN (dilution ratio 1: 1000) and phosphorylated AMPK (dilution ratio 1: 200) ) Was kept at 4 ° C. for 16 hours. After washing sufficiently, Alexa488 and Cy3 were each diluted 1: 1000 with secondary antibodies, and then washed at room temperature for 1 hour. Subsequently, fluorescence expression was photographed with a laser scanning confocal microscope.

The results of immunoblotting and immunofluorescence are shown in FIGS. 1 and 2. 1 and 2, the level of phosphorylated AMPK was increased in the Avn-C treated group according to the present invention than the vehicle-treated control group.

Example 2. Confirmation of AMPK α-adrenergic receptor (α-AR) binding degree

It was confirmed that the AMPK activity of the brain is mainly regulated by the α-adrenergic receptor (α-AR) and that the catecholamine ligand of α-AR has structural similarity to Avn-C. Accordingly, the present inventors hypothesized that AMPK will be activated by Avn-C binding to α-AR to investigate the mechanism for activation of AMPK.

Specifically, in order to confirm that Avn-C binds to α-ARs (α-1A and α-2A), Avn indicated as biotin resin on mouse hippocampal sections of the untreated and phenoxybenzamine-treated groups -C was pretreated.

After the treatment, the hippocampus sections were homogenized using a lysis buffer containing 25 mM Tris (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 1 mM NaF, and then protein It was mixed with a degrading enzyme inhibitor.

The lysate mixed with the protease inhibitor was centrifuged at 11000xg for 15 minutes at 4 ° C to remove cell nuclei and cell debris. Total protein concentration was confirmed by BCA assay (Pierce). Then, 100 μL of streptavidin beads (Thermo Scientific Inc.) was added to 600 μg of the protein lysate and spun at 4 ° C for 2 hours. The samples were washed 5 times with wash buffer (25 mM Tris pH 7.6, 150 nM NaCl, 0.5% Triton X-100), and the beads were settled after each washing with gentle centrifugation. The bound proteins were diluted twice with SDS reducing buffer and then gently heated at 60 ° C for 30 minutes. The supernatant is transferred to a new tube and heated at 90 ° C for 5 minutes before loading onto the gel.

Then, Avn-C confirmed α-1A and α-2A adrenaline receptor binding through Western blotting and is shown in FIG. 3.

As shown in FIG. 3, by confirming that a band indicating that Avn-C labeled with biotin binds to α-1A appears, it shows that Avn-C can induce activation of AMPK by binding to α-1A.

Example 3. Confirmation of AMPK activation by Avn-C and α-adrenergic receptor binding

To confirm AMPK activation by Avn-C and α-adrenergic receptor binding, the active forms of IKK, NF-κB-p65, GSK3β (Ser9), and caspase-3, which are active foam-changing factors by activation of AMPK Was confirmed by analyzing the concentration and confirmation through immunoblotting.

Specifically, the hippocampal tissue lysate used for immunoblotting is RIPA buffered in hippocampal slices (Tg2576 mice and 5XFAD mice respectively) treated with avenanthramide C in each group not treated or treated with phenoxybenzamine and homogenized on ice. After pulverization with a (homogenizer), RIPA [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM PMSF (phenylmethylsulfonyl fluoride) at 4 ° C for 30 minutes. ), 1 mM DTT, and 2 μg of leupeptin and aprotinin, respectively. The crushed cells were centrifuged at 12,000 g at 4 ° C. for 20 minutes, and the resulting soluble fraction was denatured in Laemmli buffer and SDS-PAGE was performed. Protein concentration is confirmed using BCA analysis. Proteins were separated on 10% -12% gels and transferred to PVDF membranes (Millipore, Bedford, MA, USA).

The membrane was blocked with amresco, a rapid block solution solution (pH 7), and blotting was performed using enhanced chemiluminescence (ECL). Blotting includes anti-AMPK, anti-phospho-AMPK, anti-caspase-3 (1: 1000 dilution), anti-cleaved caspase-3 (1: 500), anti-NF-κB-p65 (1: 1000), anti-phospho-NF-κB-p65 (1: 1000), anti-phospho-IKKα / β (1: 1000), anti-GSK-3α / β (1: 1000), anti-phospho-GSK-3β (Ser9 ) (1: 1000), and anti-β-actin (1: 1000) antibodies were used.

As a result, as shown in FIGS. 4 and 5, the factors were not activated in Tg2576 and 5XFAD mice pretreated with phenoxybenzamine, but p-AMPK and p-GSK3β (in Tg2576 and 5XFAD mice not pretreated with phenoxybenzamine). Ser9) levels were increased, and p-IKK, cleaved caspase-3, and p-p65 levels were decreased.

It can be confirmed that AMPK is activated by increasing and decreasing the levels of the above factors, and these results suggest that Avn-C can activate AMPK by binding to α-1A.

Claims (5)

A method of preventing or treating diabetes or obesity comprising the step of administering avenanthramide C (Avn-C) extracted from oats to animals other than humans.
A pharmaceutical composition for the prevention or treatment of diabetes or obesity, comprising avenanthramide C (Avn-C) extracted from oats as an active ingredient.
According to claim 2,
Abenanthramide C is a pharmaceutical composition for preventing or treating diabetes or obesity, which activates AMP-activated protein kinase (AMPK).
A health functional food for preventing or improving diabetes or obesity, which contains abenanthramide C extracted from oats as an active ingredient.
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