KR102600785B1 - A Composition for Suppressing Appetite Comprising Phenolic Acid Derivatives as Active Ingredients - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating bulimia and a functional food composition for suppressing appetite, comprising P-coumaric acid (para-coumaric acid) or a derivative thereof as an active ingredient. The composition of the present invention induces a significant decrease in appetite by inhibiting AMPK activity in the central nervous system, and can be used as a treatment for myogenic metabolic diseases by reducing eating itself, unlike promoting lipid metabolism in peripheral tissues. The present invention is also a naturally derived compound and has few side effects even when administered for a long period of time, so it can be effectively used as an efficient treatment composition for chronic metabolic diseases.

Description

A composition for suppressing appetite comprising a phenolic acid derivative as an active ingredient {A Composition for Suppressing Appetite Comprising Phenolic Acid Derivatives as Active Ingredients}

The present invention relates to a composition for preventing or treating appetite suppressants or hyperphagia containing a naturally occurring cinnamic phenolic acid derivative, specifically P-coumaric acid (para-coumaric acid), as an active ingredient.

AMP-activated protein kinase (AMPK) is a cellular energy sensor that plays an important role in regulating whole-body energy balance [1]. Activation of AMPK is mediated by several mechanisms and requires phosphorylation of the Thr172 residue of the catalytic α-subunit [1]. It is well known that AMPK activation in peripheral metabolic tissues such as liver, muscle, and adipose tissue improves glucose and lipid metabolism and insulin sensitivity by switching cell metabolism from an anabolic process to a catabolic process [1, 2]. However, in the central nervous system, activation of AMPK in the hypothalamus causes eating and weight gain, whereas inhibition of AMPK activity in the hypothalamus leads to anorexia, weight loss, and reduced glucose production [3-5]. Regulation of AMPK activity in the hypothalamus mediates the effects of leptin on food intake and whole-body energy metabolism [3, 6]. Due to the multifaceted actions of AMPK on metabolic activity, it has been a promising target for the treatment of obesity and metabolic diseases [7-9].

AMPK activity can be modulated by clinical therapeutic agents such as metformin and some natural plant ingredients [10]. Despite their diverse structures, most of these natural compounds can be classified as polyphenols, which are abundant in plants and have diverse biological activities such as antioxidant, anti-inflammatory, antibacterial, antidiabetic, and antiproliferative activities [10, 11]. Among natural polyphenols, resveratrol and some flavonoids have been studied for their ability to activate the AMPK pathway and improve metabolism [12], but there has been little research on the AMPK activation mechanism and metabolic effects of phenolic acid, a non-flavonoid polyphenol.

Numerous papers and patent documents are referenced and citations are indicated throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly explain the content of the present invention and the level of technical field to which the present invention pertains.

Non-patent literature 1. Seon-A Yoon et al., Biochemical and Biophysical Research Communications 432:553-557 (2003)

The present inventors have made extensive research efforts to develop an efficient natural therapeutic agent suitable for the treatment of chronic diseases, which has excellent therapeutic activity against various metabolic diseases such as obesity, diabetes, dyslipidemia, and fatty liver, and has minimal side effects even when administered for a long period of time. . As a result, it was discovered that a cinnamic phenolic acid derivative compound represented by Formula 1 below induces a decrease in eating through a significant decrease in appetite by inhibiting the activity of AMPK (AMP-activated protein kinase) in the central nervous system. By doing so, the present invention was completed.

Therefore, an object of the present invention is to provide a composition for preventing or treating bulimia.

Another object of the present invention is to provide a functional food composition for suppressing appetite.

Another object of the present invention is to provide a functional food composition for inhibiting AMPK (AMP-activated protein kinase) activity in the hypothalamus.

Other objects and advantages of the present invention will become clearer from the following detailed description, claims, and drawings.

According to one aspect of the present invention, the present invention provides a composition for preventing or treating bulimia, comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:

Formula 1

In Formula 1, R ₁ and R ₂ are each independently selected from the group consisting of hydrogen, hydroxy, and C ₁ -C ₂ alkoxy.

The present inventors have made extensive research efforts to develop an efficient natural therapeutic agent suitable for the treatment of chronic diseases, which has excellent therapeutic activity against various metabolic diseases such as obesity, diabetes, dyslipidemia, and fatty liver, and has minimal side effects even when administered for a long period of time. . As a result, it was discovered that the cinnamic phenolic acid derivative compound represented by Formula 1 induces a decrease in eating through a significant decrease in appetite by inhibiting the activity of AMPK (AMP-activated protein kinase) in the central nervous system. did.

As used herein, the term “bulimia” is one of the eating disorders and refers to a disease in which control over eating is lost due to abnormalities in the central nervous system and excessive food intake continues for a long period of time. The present inventors have discovered that P-coumaric acid, which is known to activate AMPK in peripheral organs, suppresses the activity of AMPK in the hypothalamus, thereby improving the peripheral metabolism of already ingested lipids and sugars, as well as reducing food intake by reducing appetite. It was newly discovered that it significantly reduces . Therefore, the composition of the present invention can be usefully used in the treatment of bulimia nervosa, which is a condition in which uncontrolled excessive eating continues due to abnormalities in the central nervous system while showing normal glucose and lipid metabolism, and obesity resulting therefrom. .

Accordingly, the term “composition for preventing or treating hyperphagia” in this specification has the same meaning as “appetite suppressant (Anorexiant).”

According to a specific embodiment of the present invention, hyperphagia that can be prevented or treated with the composition of the present invention is selected from the group consisting of polyphagia, excessive hunger, and hyperleptinemia.

As used herein, the term “prevention” refers to suppressing the occurrence of a disease or disease in a subject who has not been diagnosed as having the disease or disease but is likely to develop the disease or disease.

As used herein, the term “treatment” refers to (a) inhibiting the development of a disease, condition or symptom; (b) alleviation of a disease, condition or symptom; or (c) means eliminating a disease, condition or symptom. When the composition of the present invention is administered to a subject, it suppresses the activity of AMPK in the hypothalamus, strengthens leptin signaling, enhances the expression of Pomc, an appetite-suppressing neuropeptide, and suppresses the expression of Agrp, an appetite-enhancing neuropeptide, thereby suppressing abnormal appetite. It plays a role in suppressing the development of diseases caused by direct or indirect causes, eliminating them, or alleviating them. Accordingly, the composition of the present invention may be used as a treatment composition for hyperphagia by itself, or may be administered together with other pharmacological ingredients and applied as an adjuvant for the treatment of hyperphagia. Accordingly, in this specification, the term “treatment” or “therapeutic agent” includes the meaning of “therapeutic aid” or “therapeutic aid.”

As used herein, the term “administration” or “administer” refers to directly administering a therapeutically effective amount of the composition of the present invention to a subject so that the same amount is formed in the subject's body.

In the present invention, the term “therapeutically effective amount” refers to the content of the composition in which the pharmacological ingredients in the composition are contained in a sufficient amount to provide a therapeutic or preventive effect to the individual to whom the pharmaceutical composition of the present invention is to be administered. It is meant to include a “prophylactic effective amount.”

As used herein, the term “subject” includes, without limitation, humans, mice, rats, guinea pigs, dogs, cats, horses, cattle, pigs, monkeys, chimpanzees, baboons, or rhesus monkeys. Specifically, the subject of the present invention is a human.

As used herein, the term “pharmaceutically acceptable salt” includes salts derived from pharmaceutically acceptable inorganic acids, organic acids, or bases. Examples of suitable acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, trifluoroacetic acid, citric acid, methane. Examples include sulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, and benzenesulfonic acid. Salts derived from suitable bases may include alkali metals such as sodium, alkaline earth metals such as magnesium, ammonium, and the like.

As used herein, the term “alkyl” refers to a straight-chain or branched saturated hydrocarbon group and includes, for example, methyl, ethyl, propyl, isopropyl, etc. C ₁ -C ₃ alkyl refers to an alkyl group having an alkyl unit having 1 to 3 carbon atoms, and when C ₁ -C ₃ alkyl is substituted, the carbon number of the substituent is not included.

According to a specific embodiment of the present invention, R ₁ and R ₂ in Formula 1 are hydrogen and hydroxy, respectively. The compound of Formula 1 in which R ₁ and R ₂ are hydrogen and hydroxy, respectively, is P-coumaric acid (para-coumaric acid, C ₉ H ₈ O ₃ , (E)-3-(4-hydroxyphenyl-2-phorophene) P-coumaric acid is a natural phenolic acid compound expressed in various plants. It is known to have antioxidant, anti-inflammatory and anti-cancer activities and to inhibit lipid accumulation by activating AMPK in the liver and skeletal muscles, but it is not known in these peripheral organs. Unlike this, the present inventors first discovered that AMPK activity is inhibited in the central nervous system, such as the hypothalamus.

According to a specific embodiment of the present invention, the composition of the present invention inhibits the activity of AMPK (AMP-activated protein kinase) in the hypothalamus.

As used herein, the term “inhibition of activity” means causing a decrease in the activity or expression of the target protein, which not only causes the activity or expression of the target protein to become undetectable or exists at an insignificant level, but also refers to the case where the activity or expression of the target protein becomes undetectable or exists at an insignificant level. This means lowering the activity or expression of AMPK to the extent that its biological function (i.e., the function as a phosphorylating enzyme of AMPK) can be significantly inhibited.

According to a specific embodiment of the present invention, the composition of the present invention enhances leptin signaling in the hypothalamus.

When the composition of the present invention is prepared as a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are those commonly used in preparation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, Includes, but is limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. It doesn't work. In addition to the above ingredients, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered orally or parenterally, specifically, orally, intravenously, subcutaneously, or intraperitoneally.

The appropriate dosage of the pharmaceutical composition of the present invention is prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity. It can be. The preferred dosage of the pharmaceutical composition of the present invention is within the range of 0.001-100 mg/kg for adults.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating it using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by a person skilled in the art. Alternatively, it can be manufactured by placing it in a multi-capacity container. At this time, the formulation may be in the form of a solution, suspension, syrup or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, powder, granule, tablet or capsule, and may additionally contain a dispersant or stabilizer.

According to another aspect of the present invention, the present invention provides a functional food composition for suppressing appetite comprising a compound represented by the following formula (1) or a foodologically acceptable salt thereof as an active ingredient:

Formula 1

Since the compound of Formula 1 used in the present invention and its appetite suppressing function have already been described in detail, their description is omitted to avoid excessive duplication.

As used herein, the term "foodologically acceptable salt" refers to a salt in a form that can be used in food compositions among salts in which cations and anions are combined by electrostatic attraction, and specific examples thereof include the above-mentioned "pharmaceutically acceptable salt." Includes examples of “salts that become salts.”

According to another aspect of the present invention, the present invention provides a functional food composition for inhibiting the activity of AMPK (AMP-activated protein kinase) in the hypothalamus, comprising a compound represented by the following formula (1) or a food-acceptable salt thereof as an active ingredient: Provides:

Formula 1

Since the compound of Formula 1 used in the present invention and its function to inhibit AMPK activity in the hypothalamus have already been described in detail, the description is omitted to avoid excessive duplication.

When the composition of the present invention is manufactured as a food composition, it may contain not only the compound of the present invention as an active ingredient, but also carbohydrates, seasonings, and flavoring agents commonly added during food production. Examples of carbohydrates include monosaccharides such as glucose and fructose; It includes, but is not limited to, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As a flavoring agent, natural flavoring agents (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is manufactured as a drink, in addition to the pine bark extract, which is the active ingredient of the present invention, citric acid, high fructose corn syrup, sugar, glucose, acetic acid, malic acid, fruit juice, jujube extract, jujube extract, licorice extract, etc. are added. It can be included as .

According to another aspect of the present invention, the present invention provides an appetite suppressant (Anorexiant) comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:

Formula 1

In Formula 1, R ₁ and R ₂ are each independently selected from the group consisting of hydrogen, hydroxy, and C ₁ -C ₂ alkoxy.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a pharmaceutical composition for preventing or treating bulimia and a functional food composition for suppressing appetite, comprising P-coumaric acid (para-coumaric acid) or a derivative thereof as an active ingredient.

(b) The composition of the present invention induces a significant decrease in appetite through inhibition of AMPK activity in the central nervous system, and can be used as a treatment for myogenic metabolic diseases by reducing eating itself, unlike promoting lipid metabolism in peripheral tissues.

(c) The present invention is also a naturally derived compound and has few side effects even when administered for a long period of time, so it can be effectively used as an efficient treatment composition for chronic metabolic diseases.

Figure 1 is a diagram showing the different effects of CA on AMPK activation in HepG2 hepatocytes and hypothalamic N1 cells, respectively. Figures 1A and 1B are immunoblotting results (Figure 1A) and quantitative graphs (Figure 1B) showing the effect of treatment with CA and metformin on AMPK activation in HepG2 hepatocytes. Figures 1c and 1d are immunoblotting results (Figure 1c) and quantitative graphs (Figure 1d) showing the effect of treatment with CA and metformin on AMPK activation in N1 cells of the hypothalamus. Each result was expressed as mean ± standard error. Two-tailed Student's t -test. *P<0.05.
Figure 2 is a picture showing the different effects of CA on AMPK activation in vivo . Figures 2A and 2B are immunoblotting results (FIG. 2A) and quantitative graphs (FIG. 2B) showing the effect of treatment with CA and metformin on AMPK activation in mouse liver. Figures 2c and 2d are immunoblotting results (Figure 2c) and quantitative graphs (Figure 2d) showing the effect of treatment with CA and metformin on AMPK activation in the hypothalamus. Figures 2e and 2f are immunoblotting results (Figure 2e) and a quantitative graph (Figure 2f) showing the effect of treatment with CA and metformin on p70S6K activation in the hypothalamus. Each result was expressed as mean ± standard error. Multiple groups were compared using one-way analysis of variance and Tukey's post hoc test or Student's t-test. *P < 0.05, **P < 0.01.
Figure 3 is a diagram showing that leptin sensitivity in the hypothalamus is enhanced by CA treatment. Figure 3A shows the effect of vehicle (veh), CA or metformin treatment on HFD-induced hyperleptinemia. ***P < 0.001 compared to NC group, #P < 0.05 compared to HFD-Veh group. Figures 3b and 3c are immunoblotting results (Figure 3b) and quantitative graphs (Figure 3c) showing the effect of treatment with CA and metformin on STAT3 downstream signaling in the hypothalamus. Figure 3d shows the results of measuring the transcription of Pomc, Agrp, and Npy in hypothalamic N1 cells under CA treatment. Figure 3e shows the results of measuring daily food intake of HFD-fed mice treated with vehicle (veh) or p-coumaric acid (CA) (3-day average). Each result was expressed as mean ± standard error. Multiple groups were compared using one-way analysis of variance and Tukey's post hoc test or Student's t-test. *P<0.05, ***P<0.001.
Figure 4 is a diagram showing that CA treatment improves whole body glucose homeostasis in mice. Figure 4a shows the results of measuring body weight of NC-diet and HFD-diet mice administered vehicle (veh), metformin (met), or p-coumaric acid (CA) for 8 weeks. Figure 4b shows the results of measuring fasting blood sugar levels in NC-diet and HFD-diet mice administered vehicle (veh), metformin (met), or p-coumaric acid (CA) for 8 weeks. Figure 4c shows the results of measuring plasma insulin levels in NC-fed and HFD-fed mice administered vehicle (veh), metformin (met), or p-coumaric acid (CA) for 8 weeks. Figures 4d and 4e show GTT (D) and GTT AUC (E) measured in NC-fed and HFD-fed mice administered vehicle (veh), metformin (met), or p-coumaric acid (CA) for 8 weeks. It is a result. Figures 4f and 4g show ITT (F) and ITT AUC (G) measured in NC-diet and HFD-diet mice administered vehicle (veh), metformin (met), or p-coumaric acid (CA) for 8 weeks. It is a result. Each result was expressed as mean ± standard error. One-way analysis of variance and Tukey's post-hoc test were displayed as bar graphs, and two-way analysis of variance and Bonferroni's post-hoc test were displayed as line graphs. *P < 0.05, **P < 0.01, ***P < 0.001 compared to NC group. #P < 0.05, ##P < 0.01 compared to HFD-Veh group.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Experiment method

experiment material

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and penicillin-streptomycin (P/S) were purchased from Gibco (Life Technologies, NY, USA). P-Coumaric acid (Cat. No C9008) was purchased from Sigma (St. Louis, MO, USA), and metformin was purchased from TOKU-E (Bellingham, WA, USA). Antibodies AMPKα, antibodies against p-AMPKα (Thr172) were purchased from Cell Signaling Technology (Beverly, MA, USA). Protease inhibitor tablets were purchased from Thermo Fisher Scientific (Thermo Fisher Scientific Inc., MA, USA), and phosphatase inhibitor cocktails were purchased from Roche (F. Hoffmann-La Roche Ltd., Basel, Switzerland). All other reagents were purchased from Intron Biotechnology (Intron Biotechnology Co., Ltd., Gyeonggi-do, Korea) unless otherwise noted. The storage and use solutions of metformin were prepared by dissolving in water. A stock solution of p-coumaric acid was prepared in dimethylsulfoxide (DMSO). The p-coumaric acid solution was diluted with DMSO and used for cell culture, or diluted with PBS (phosphate buffer saline) (1/20, v/v) and used for oral administration in animal experiments.

Cell culture and treatment of compounds

HepG2 hepatocytes and hypothalamic N1 cells were maintained in DMEM medium containing 10% FBS and 1% P/S and cultured in a humidified environment at 5% CO ₂ and 37°C. Cells were treated with metformin dissolved in water at a final concentration of 2 μM or p-coumaric acid dissolved in DMSO at a final concentration of 20 μM, and the cells were collected, lysed, and measured for mRNA and protein levels by real-time qPCR or Western blot.

laboratory animals

All animal experiments were performed under the approval of the Laboratory Animal Ethics Committee (IACUC) of Tan Tao Medical University (43/HDKH.TTU.2019). Swiss albino male mice (4 weeks old) were purchased from Pasteur (Ho Chi Minh city, Viet Nam). Mice were maintained at room temperature (22-25°C) under a 12-hour light/dark cycle (lights on/off at 06:00 a.m./p.m.). Mice were fed a regular diet (AniFood, Pasteur Institute-VN, 3.84 kcal/kg, 6-8% kcal from fat, NC) or a high-fat diet (Research Diets D12492, USA, 5.24 kcal/kg, 60% kcal from fat, HFD). ) were allowed to eat freely. After 1 week of adaptation, mice were divided into four experimental groups: NC, HFD-Veh, HFD-Met, and HFD-CA (6 mice per group). The NC group was fed normal diet and purified water. The HFD-CA group was fed HFD and administered p-coumaric acid (200 mg/kg) orally daily for 8 weeks. HFD-Veh was used as a vehicle control group by orally administering the same volume of DMSO/PBS mixed solution for the same period of time. HFD-Met was used as standard treatment by administering metformin (200 mg/kg) dissolved in water while feeding HFD.

Blood glucose levels were measured as indicated, and glucose and insulin resistance tests (GTT and ITT) were performed 6 weeks after administration. At the end of the experiment, mice were sacrificed and organs were collected and stored at -80°C until further analysis.

Dissection and collection of hypothalamic tissue

Mice were reared under normal dietary conditions and sacrificed after anesthesia with ketamine (100 mg/kg body weight). After skull removal, intact brains were collected and washed with ice-cold 1 x phosphate buffer saline (PBS). The hypothalamic region was observed under a low-power dissecting microscope and dissected from the entire brain tissue. All hypothalamic samples were immediately rapidly cooled on dry ice and stored at -80°C.

Food intake measurement

Experimental mice were individually housed, and daily food intake from 8:00 to 15:00 was recorded for 3 days and normalized to initial body weight.

Insulin and leptin measurements

Blood samples were collected from tail-nick blood drops, and insulin and leptin were measured using an ELISA kit (Morinaga Institute of Biological Science, Yokohama, Japan) according to the manufacturer's instructions.

GTT and ITT experiments

GTT was performed using a previously reported method [34]. Briefly, mice were starved for 18 hours and drinking water was available ad libitum. After measuring fasting blood sugar, glucose (1.2 g/kg body weight) was administered intraperitoneally, and blood was collected from the tail 15, 30, 60, 90, and 120 minutes later. For ITT, mice were starved for 2 hours and drinking water was available ad libitum. After measuring basal blood sugar, 1U/kg body weight of short-acting insulin (Eli Lilly and Company, IN, USA) was administered intraperitoneally, and blood glucose levels were monitored at regular time intervals. Blood glucose levels were measured using the glucose oxidase method using a commercially available blood glucose meter (SAFE-ACCU, Shanghai International Holding Corp., Germany).

SDS-PAGE and Western blotting

Cells were washed with 1×PBS and lysed with RIPA buffer containing protease and phosphatase inhibitors (Thermo Fisher Scientific Inc., MA, USA, Cat. No. 89900). For in vivo experiments, tissues were homogenized with a Dounce homogenizer and lysed with RIPA buffer containing protease and phosphatase inhibitors. The total protein concentration in the dissolved sample was measured using the PierceTM Coomassie (Bradford) Protein Assay kit (Thermo Fisher Scientific Inc., MA, USA). After SDS-PAGE, Western blotting was performed and the Miracle StarTM Femto Western blot detection system (Intron Biotechnology Co., Ltd., Korea) and X-ray film (UltraCruz® Autoradiography Film, Santa Cruz Biotechnology Inc., TX, USA) were used. The blot was visualized using Quantification of blots was performed using NIH ImageJ software.

AMPKα (Cat. No. 2532, dilution 1:5,000), phosphor AMPKα Thr172 (Cat. No. 2531, dilution 1:5,000), p70 S6 (Cat. No. 9202, dilution 1:5,000), phosphor-p70 S6 Thr389 Primary antibodies against (Cat. No. 9204, dilution 1:5,000), Stat3 (Cat#.No. 9139, 1:5,000) and phosphor-Stat3 (Tyr705) (Cat#.No. 9131, 1:5,000) was purchased from Cell Signaling (Cell Signaling Technology Inc., MA, USA). Antibody against GAPDH (Cat. No. GTX100118, dilution 1:10,000) was purchased from GeneTex (GeneTex Inc., CA, USA). Primary antibodies were prepared in 3% bovine serum albumin in Tris buffered saline (TBS) containing 0.1% (v/v) Tween 20 (TBST) and 0.05% (m/v) sodium azide. Horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody was purchased from Thermo Scientific (Thermo Fisher Scientific Inc., MA, USA) and diluted 10,000-fold in 3% nonfat dried milk in TBST.

RNA isolation and real-time qPCR

Hypothalamic N1 cells were cultured with p-coumaric acid at a final concentration of 10 mM for 24 hours. Afterwards, cells were collected, lysed, and total RNA was extracted with Ambion® Trizol reagent (Life Technologies, CA, USA). cDNA was synthesized from 1 μg of total RNA using a High-Capacity cDNA reverse transcription kit (Applied Biosystem, Warrington, UK) according to the manufacturer's instructions. For real-time PCR, cDNA and primers were prepared using Power SYBR® Green PCR Master Mix (Applied Biosystem, Warrington, UK) according to the instructions. Primer sequences used for real-time Q-PCR were as follows: Pomc, forward, 5′-CAGGTCCTGGAGTCCGAC-3′, reverse, 5′- CATGAAGCCACCGTAACG-3′; Agrp, forward, 5'-CGGCCACGAACCTCTGTAG-3', reverse, 5'-CTCATCCCCTGCCTTTTGC-3'; Npy, forward, 5'-CTACTCCGCTCTGCGACACT-3', reverse, 5'-AGTGTCTCAGGGGCTGGATCTC-3'; and 18S, forward, 5’-AACCCGTTGAACCCCATT-3’, reverse, 5’-335 CCATCCAATCGGTAGTAGCG-3’.

statistical analysis

All statistical analyzes were performed using Prism 5.0 software. Body weight GTT and ITT at each time point were analyzed using two-way analysis of variance. One-way analysis of variance was used to analyze blood glucose levels, plasma metabolic hormone levels, and food intake in the different treatment groups. Student's t-test was applied to other data. Statistical significance was considered if P < 0.05.

Experiment result

p-Coumaric acid shows different effects on peripheral and central AMPK activation

p-Coumaric acid (CA) or plant extracts containing CA have been shown to activate the AMPK pathway in several metabolic cells [16-18]. Using metformin, a standard AMPK activator, we consistently observed strong activation of AMPK when liver-derived HepG2 cells were treated with CA (Figures 1a and 1b). However, surprisingly, the present inventors found that CA significantly inhibited AMPK activation in N1 cells of the hypothalamus, which was similar to the previously reported action of metformin (Figures 1c and 1d) [19].

Next, to further confirm the effect of CA on AMPK activation in vivo , CA or metformin was orally administered to mice at a dose of 200 mg/kg body weight, and AMPK activation in metabolic organs including the liver and hypothalamus was examined. Similar to the results observed in cell lines, AMPK was confirmed to be significantly activated in mouse liver by CA treatment (Figures 2a and 2b). On the other hand, AMPK activity in hypothalamic samples was significantly inhibited by CA treatment (Figures 2c and 2d). Taken together, we can see that CA treatment activates AMPK in the liver and inhibits AMPK in the hypothalamus both in vivo and in vitro .

Next, we sought to investigate the molecular mechanism by which CA inhibits AMPK activity in the center. S6 kinase forms a complex with AMPK and reduces AMPK activity by inducing phosphorylation of the ser491 residue [6, 20]. Furthermore, it has been reported that metformin activates S6 kinase and inhibits AMPK activity in the hypothalamus [21]. Accordingly, the present inventors hypothesized that CA treatment would also be able to regulate AMPK activation in the hypothalamus through regulation of S6 kinase activity. In fact, the phosphorylated form of p70 S6 kinase in the hypothalamus of CA- or metformin-treated mice was significantly increased, indicating that S6 kinase was activated by CA treatment (Figures 2e and 2f). Through this, it was found that CA inhibits AMPK activity in the hypothalamus by enhancing S6 kinase activity.

p-Coumaric acid treatment enhances leptin signaling in the hypothalamus

AMPK and S6 kinases in the hypothalamus are known to regulate food intake in response to leptin-melanocortin signaling [3]. Furthermore, metformin treatment enhances leptin receptor signaling in the hypothalamus and restores leptin sensitivity in HFD-induced obese rodents with leptin resistance [22, 23]. Accordingly, the present inventors hypothesized that CA treatment would have affected leptin signaling in the functional hypothalamus and suppressed food intake. To investigate this, we induced leptin resistance using a HFD-induced obesity mouse model and then examined the effect of CA on leptin signaling in the hypothalamus. As shown in Figure 3A, the plasma leptin level of HFD-fed mice was significantly increased compared to that of normal-fed mice, leading to leptin resistance. Consistent with previous findings, metformin treatment improved the hyperleptinemic state and increased leptin signaling in the hypothalamus by increasing the phosphorylated form of signal transducer and activator of transcription 3 (STAT3), a well-known downstream effector of leptin signaling in the hypothalamus. was strengthened (Figures 3a-3c) [24, 25]. In particular, CA treatment improved HFD-induced hyperleptinemia and increased hypothalamic p-STAT3 levels (Figures 3A-3C). In addition, CA treatment enhances the expression of the appetite suppressing neuropeptide proopiomelanocortin (Pomc) while suppressing the expression of the orexigenic agouti-related neuropeptide (Agrp), which is a hypothalamic leptin-related neuropeptide. It is a well-known target gene of STAT3 signaling [26]. Accordingly, CA-treated mice had reduced intake of high-fat diet compared to control vehicle-treated mice (Figure 3e). These results clearly show that CA treatment enhances functional leptin signaling in the hypothalamus.

p-Coumaric acid treatment improves systemic glucose homeostasis

Based on the effects of CA on peripheral and central AMPK activation, hypothalamic leptin signaling, and food intake, we hypothesized that CA treatment would regulate body weight and glucose homeostasis. To confirm this, the metabolic effects of CA treatment were further investigated using a HFD-induced mouse model. HFD-diet mice were orally administered CA (200 mg/kg body weight) or vehicle for 8 weeks, and HFD-diet mice were orally administered metformin (200 mg/kg body weight) as standard treatment, while NC-diet mice were administered orally for 8 weeks. It was used as a negative control. Contrary to expectations, there was no statistically significant difference in body weight between vehicle and CA-treated HFD-fed mice, with only a slight trend toward weight loss in CA-treated mice, especially during the first 2 weeks after HFD feeding (Figure 4a). However, CA-treated mice had a significant decrease in fasting blood glucose (Figure 4b) and decreased plasma insulin (Figure 4c). These results showed that blood sugar was better controlled in CA-treated mice than in the vehicle control group. Therefore, next, glucose and insulin resistance tests (GTT and ITT) were performed to further investigate the effect of CA treatment on glucose resistance and insulin sensitivity. As shown in Figures 4d and 4e, CA-treated mice were found to have higher glucose resistance as intraperitoneally administered glucose was well eliminated in the GTT test. Furthermore, these mice also showed a significant increase in endogenous glucose clearance per hour of insulin injection during ITT (Figures 4f and 4g). Taken together, it can be seen that CA treatment significantly improves insulin sensitivity and whole-body glucose homeostasis in mice.

As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

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<110> Industry-academic cooperation foundation yonsei university <120> A Composition for Suppressing Appetite Comprising Phenolic Acid Derivatives as Active Ingredients <130>HPC-9792 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Pomc F primer <400> 1 caggtcctgg agtccgac 18 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Pomc R primer <400> 2 catgaagcca ccgtaacg 18 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Agrp F primer <400> 3 cggccacgaa cctctgtag 19 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Agrp R primer <400> 4 ctcatcccct gcctttgc 18 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Npy F primer <400> 5 ctactccgct ctgcgacact 20 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Npy R primer <400> 6 agtgtctcag ggctggatct c 21 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> 18S F primer <400> 7 aacccgttga accccatt 18 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 18S R primer <400> 8 ccatccaatc ggtagtagcg 20

Claims (11)

delete delete delete delete delete A functional food composition for suppressing appetite comprising a compound represented by the following formula (1) or a foodologically acceptable salt thereof as an active ingredient:
Formula 1

In Formula 1, R ₁ is hydrogen and R ₂ is hydroxy.
delete delete delete delete delete