KR20200007990A - Composition for preventing, treating or improving obesity comprising Jaceosidin - Google Patents

Abstract

The present invention relates to a composition for preventing, treating or alleviating obesity containing jaceosidin as an active component. The jaceosidin has an effect of inhibiting differentiation of adipocytes and reducing lipid accumulation in cells. In addition, the jaceosidin exhibits an excellent anti-obesity effect by inhibiting the expression of peroxysome proliferator activator receptor gamma (PPARγ) and CCAAT-enhancer binding protein alpha (C/EBPa) and adiponectin, which are transcription factors involved in adipocyte formation and differentiation. Therefore, jaceosidin of the present invention can be usefully used as a pharmaceutical composition and health functional food for preventing, treating or alleviating obesity.

Description

Composition for preventing, treating or improving obesity comprising Jaceosidin

The present invention relates to a composition for preventing, treating, or improving obesity, comprising asceodine as an active ingredient.

Obesity generally means that the body has an excessive amount of fat tissue, and the energy consumed by food is not balanced with the energy consumed by physical activity, so that surplus energy is accumulated as body fat. Obesity is caused by a wide variety of causes, including genetic factors, irregular eating habits, lack of exercise, or environmental effects from Western diets, depression, boredom, psychological effects from excessive stress, and pathological factors caused by thyroid or corticosteroid changes. It is known to be caused by. Recently, many changes have been made to eating habits due to economic growth and lifestyle changes, and the busy modern people are increasing overweight and obesity due to high calorie diets and fast exercise.

In addition, since obesity is caused by the enlargement of adipocytes, inhibition of adipocyte differentiation may be helpful in the treatment of obesity (Henninger, Eliasson, Jenndahl, & Hammarstedt, 2014). In the past, anti-obesity studies related to the inhibition of adipocyte differentiation and lipid accumulation have been reported (Kim & Lee, 2017; Li et al., 2017), where 3T3-L1 mouse fibroblasts are responsible for the differentiation of adipocyte progenitors. A well-characterized model for this is commonly used in in vitro studies (Kaestner et al., 1989; SK Kim & Kong, 2010).

Obesity is perceived more seriously because of the many complications that can be caused by obesity than by its own risk. If body fat is abnormally increased due to energy imbalance over time, diabetes, hyperlipidemia, heart disease, stroke, arteriosclerosis And various metabolic diseases such as fatty liver and adult disease. In addition, obesity can cause not only physical illness but also mental illness such as social isolation, alienation, lack of self-confidence and depression, which is a serious social problem not only in the West but also in Korea, and the need for prevention and treatment of obesity is very important. Is recognized.

Obesity can be treated through diet, regular exercise, lifestyle improvements such as behavioral therapy, and drugs such as appetite suppressants and fat absorption inhibitors. Obesity is a chronic disease that requires long-term use when attempting drug treatment. Currently, products approved for long-term use in Korea for more than three months include sibutramine, an appetite suppressant, and orlistat, an lipase inhibitor. There is). However, most of these anti-obesity drugs are psychotropic drugs that act on the central nervous system to regulate appetite, so they have side effects such as headache and vomiting, and there are problems such as abuse concerns. Therefore, studies are being actively conducted to develop materials having high stability and excellent anti-obesity effect that can solve the side effects of the commercially available anti-obesity agents.

Meanwhile, jasosidin is Artemisia The main flavonoid compound of the genus is known to be contained in large amounts in mugwort and wormwood. It has been reported that zodiacic acid has several biological effects, including anti-tumorogenesis, antioxidant, and anti-inflammatory effects (Khan, Rasul, Yi, Zhong, & Ma, 2011; Min, Kim, Baek, & Kim, 2009). No anti-obesity effect has been reported yet.

The present inventors have conventionally demonstrated the anti-lipogenic effect of hispidulin, which has a chemical structure similar to that of zithrodine (H.-S. Kim et al., 2017), and zithrodine has similar effects. As a result of assuming that it will have a study, it was confirmed that the postural oscidin has an excellent anti-obesity effect, so that it can be used as a composition for preventing, treating, or improving obesity.

The present inventors have tried to develop a composition for preventing or treating obesity, which has high stability and excellent anti-obesity effect that can solve the problems of the conventional anti-obesity agent, and confirmed that the postural osdine of the present invention had an excellent anti-obesity effect. The invention was completed.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of obesity, comprising asceodine as an active ingredient.

In addition, an object of the present invention is to provide a dietary supplement for the prevention or improvement of obesity comprising a postural osdine as an active ingredient.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention or treatment of obesity, comprising the postural osdine as an active ingredient.

In addition, the present invention provides a dietary supplement for preventing or improving obesity, comprising a postural osdine as an active ingredient.

In one embodiment of the present invention, the composition can inhibit the differentiation of adipocytes.

In another embodiment of the present invention, the composition may reduce lipid accumulation in cells.

In another embodiment of the present invention, the composition may inhibit the expression of a transcription factor or adiponectin involved in adipocyte formation and differentiation.

In another embodiment of the present invention, the transcription factor may be peroxysome proliferator activated receptor gamma (PPARγ) or CCAAT-enhancer binding protein alpha (C / EBPa).

The present invention also provides a method for preventing or treating obesity, comprising administering the composition to a subject.

In addition, the present invention provides a preventive or therapeutic use of the composition.

Postiosidine according to the present invention has the effect of inhibiting the differentiation of adipocytes and reducing the accumulation of lipids in the cells, peroxysomal proliferator activating receptor gamma (PPARγ) and CCAAT which are transcription factors involved in adipocyte formation and differentiation It was confirmed that the anti-obesity effect is excellent by inhibiting the expression of enhancer binding protein alpha (C / EBPa) and adiponectin, the postural oscidin of the present invention is a pharmaceutical composition for preventing or treating obesity, or improving health functional foods. It is expected to be used in various ways.

Figure 1a is a diagram showing the results of measurement of cell viability in 3T3-L1 adipocytes treated with postural oscillin according to an embodiment of the present invention by MTT analysis.
Figure 1b is a result of observing the cell shape of 3T3-L1 adipocytes, negative control (NC), and positive control (PC) treated with postural oscillin according to an embodiment of the present invention using a 400x magnification microscope The figure which shows.
Figure 2a is a view showing the result of Oil Red O staining in 3T3-L1 adipocytes treated with postural oscidin according to an embodiment of the present invention.
Figure 2b is a view showing the measurement of the content of Triglyceride (TG) in 3T3-L1 adipocytes treated with postural oscidin according to an embodiment of the present invention.
Figure 3a is a diagram showing the mRNA expression levels of PPARγ, C / EBPa, and adiponectin by treatment with postural osdine in 3T3-L1 adipocytes according to an embodiment of the present invention.
Figure 3b is a view showing the expression level of PPARγ and adiponectin protein by treatment with postural osdine in 3T3-L1 adipocytes according to an embodiment of the present invention.

As the present invention allows for various changes and numerous embodiments, particular embodiments will be illustrated in the drawings and described in detail in the written description. However, this is not intended to limit the present invention to specific embodiments, it should be understood to include all transformations, equivalents, and substitutes included in the spirit and scope of the present invention. In the following description of the present invention, if it is determined that the detailed description of the related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for the prevention or treatment of obesity, comprising asceodine as an active ingredient.

The obesity, which is a disease to be prevented or treated according to the present invention, refers to a state in which fat cells proliferate and differentiate due to metabolic disorders, and thus fat is excessively accumulated, and hypertension, diabetes mellitus, and dyslipidemia Associated complications, including metabolic syndrome, may be caused. When energy absorption increases relative to consumption, the mass and volume of fat cells increase, resulting in an increase in the mass of fat tissue.

As used herein, the term "prevention" means any action that inhibits obesity or delays the onset by administration of a pharmaceutical composition according to the invention.

As used herein, the term "treatment" refers to any action by which administration of the pharmaceutical composition according to the present invention improves or advantageously alters the symptoms of obesity.

The present invention also provides a method for preventing or treating obesity, comprising administering the pharmaceutical composition to a subject.

As used herein, the term "administration" means providing a subject with any composition of the present invention in any suitable manner.

As used herein, the term "individual" means a subject in need of treatment of a disease, and more specifically, human or non-human primates, mice, dogs, cats, horses, cattle, and the like. Mean mammal.

The present invention also provides a use of the pharmaceutical composition for the prevention or treatment of obesity.

The postural olidine according to the present invention may inhibit the differentiation of adipocytes and reduce lipid accumulation in adipocytes. In addition, the expression of a transcription factor or adiponectin involved in adipocyte formation and differentiation can be suppressed. At this time, the transcription factor may be, but is not limited to, peroxysome proliferator activator receptor gamma (PPARγ) or CCAAT-enhancer binding protein alpha (C / EBPa).

In one experimental example of the present invention, after the posture treatment with postural osdine during the differentiation period of 3T3-L1 adipocytes, it was observed that postural azidine strongly inhibits the differentiation of 3T3-L1 adipocytes in a concentration-dependent manner. It confirmed (see Experimental example 2 and FIG. 1b).

In another experimental example of the present invention, as a result of lipid staining in 3T3-L1 adipocytes using the Oil red O staining test technique, lipid accumulation in the 3T3-L1 adipocytes treated with postose was reduced in a concentration-dependent manner. It was confirmed that the TG content in the 3T3-L1 adipocytes treated with postural osdine was significantly reduced compared to the positive control group (see Experimental Example 3 and FIGS. 2A to 2B).

In another experimental example of the present invention, RNA synthesized from 3T3-L1 adipocytes treated with zirconia was synthesized by cDNA, followed by RT-PCR. As a result, zirconia was expressed as PPARγ, C / EBPa, and It was confirmed that the mRNA expression of adiponectin was suppressed in a concentration-dependent manner (see Experimental Example 4-1 and Figure 3a).

In addition, as a result of analyzing the protein expression in 3T3-L1 adipocytes treated with postural oscillin, it was confirmed that the postiosidine inhibits the expression of PPARγ2 and adiponectin protein (see Experimental Example 4-2 and FIG. 3B).

Differentiation of adipocytes is regulated by changes in expression of various transcription factors such as peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT-enhancer-binding protein alpha (C / EBPα) (Yoo, Seo, Shin, & Jeong, 2015 ).

At this time, the peroxisome proliferator-activated receptor gamma (PPARγ) is an important factor in the cell signal pathway and is known to regulate transcription factors related to glucose metabolism and adipocyte formation. PPARγ is expressed in the form of PPARγ1 and PPARγ2 in adipocytes. PPARγ2 has 30 more amino acids at the N-terminus than PPARγ1, but the remaining amino acid sequences are the same, and the expression levels of PPARγ1 and PPARγ2 differ from tissue to tissue. PPARγ2 is specifically expressed in adipocytes, while PPARγ1 is expressed at low levels in several cell types such as macrophages, colonic epithelial cells, and endothelial cells (Lehrke & Lazar, 2005; Sarjeant & Stephens, 2012). PPARγ2 can have a significant effect on adipogenesis and has been found in adipocytes of obese patients (Ren, Collingwood, Rebar, Wolffe, & Camp, 2002; Sarjeant & Stephens, 2012).

CCAAT-enhancer-binding protein alpha (C / EBPα) is a protein that plays a pivotal role with PPARγ in regulating the differentiation of adipocytes. In the process of differentiating progenitor cells, early stages of C / EBP and C / EBP induce the expression of PPARγ, and the increased expression of PPARγ is responsible for the late stage of differentiation of adipocytes by C / EBPa expression.

Adiponectin is also adipokine secreted by adipocytes, and has been reported to control insulin resistance-related obesity (Lindsay et al., 2002).

On the other hand, the "pharmaceutical composition" in the present invention means that it is prepared for the purpose of preventing or treating a disease, and can be used by formulating in various forms, respectively, in accordance with conventional methods. For example, it may be formulated in oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, and syrups, and may be formulated in the form of external preparations, suppositories, and sterile injectable solutions.

The pharmaceutical composition according to the present invention may include a pharmaceutically acceptable carrier in addition to the active ingredient. At this time, the pharmaceutically acceptable carrier is commonly used in the formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose , Polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. In addition to the above components, lubricants, wetting agents, sweetening agents, flavoring agents, emulsifiers, suspending agents, preservatives and the like may be further included.

The pharmaceutical compositions of the invention may be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage may be determined by the condition and weight of the patient, Depending on the degree, drug form, route of administration, and time, it may be appropriately selected by those skilled in the art.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level refers to the type of disease, severity, activity of the drug, It may be determined according to the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of treatment, factors including the drug used concurrently and other factors well known in the medical field. The pharmaceutical compositions according to the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can achieve the maximum effect with a minimum amount without side effects, which can be readily determined by one skilled in the art.

In addition, the present invention provides a health functional food composition for preventing or improving obesity containing a postural osdine as an active ingredient.

As used herein, the term "improvement" means any action that at least reduces the parameters associated with the condition being treated, for example, the extent of symptoms. At this time, the health functional food composition may be used simultaneously or separately with the agent for treatment before or after the onset of the disease for the prevention or improvement of obesity.

In the present invention, the "health food composition" is characterized in that it is formulated as one selected from the group consisting of tablets, pills, powders, granules, powders, capsules and liquid formulations, including one or more of carriers, diluents, excipients and additives. It is done.

Examples of the food which can be added to the posture osdine of the present invention include various foods, powders, granules, tablets, capsules, syrups, beverages, gums, teas, vitamin complexes, and health functional foods. As additives that may be further included in the present invention, natural carbohydrates, flavoring agents, nutrients, vitamins, minerals (electrolytes), flavoring agents (synthetic flavoring agents, natural flavoring agents, etc.), colorants, fillers, pactic acid and salts thereof, Alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, antioxidants, glycerin, alcohols, carbonation agents, and one or more components selected from the group consisting of pulp can be used. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the flavoring agent, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used.

In addition to the above, the composition according to the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents, fact acids and salts thereof, alginic acid and salts thereof, organic acids, protection Sex colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks and the like.

In addition, other compositions of the present invention may contain pulp for the production of natural fruit juices and vegetable beverages. These components can be used independently or in combination.

Specific examples of the carrier, excipient, diluent, and additives include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, erythritol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium phosphate, calcium silicate, Group consisting of microcrystalline cellulose, polyvinylchirrolidone, cellulose, polyvinylpyrrolidone, methylcellulose, water, sugar syrup, methyl hydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oil It is preferred that at least one selected from be used.

Hereinafter, preferred embodiments and experimental examples of the present invention will be described in detail with reference to the accompanying drawings. However, these Examples and Experimental Examples are only for illustrating the present invention, and the scope of the present invention will not be construed as being limited by these Examples and Experimental Examples.

Example 1 Sample Preparation and Cell Culture

Osiodine (Jaceosidin) It was purchased from Chem Faces (Cas num. 18085-97-7), dissolved in DMSO and used for experiments. 3T3-L1 adipocytes were purchased from Korea Cell Line Bank and cultured in medium with 10% NBCS added to Dulbecco's modified Eagle medium (DMEM) medium. For differentiation, cells were maintained for two days post-confluence and then stimulated for two days by treatment with adipocyte differentiation inducing substances (0.5 mM IBMX, 1 μM DEXA, 0.125 mM indomethacin, and 10 μg / mL insulin). Thereafter, only 10 μg / mL of insulin stimulation was incubated until complete differentiation into mature adipocytes. In order to observe the inhibitory effect of adipocyte differentiation, the postural oscidin was continuously treated during adipocyte differentiation at concentrations of 10, 20, and 40 μM.

Experimental Example 1. Measurement of Cell Viability

On cell viability In order to investigate the effect of postural osdine, 3T3-L1 adipocytes were seeded in a 96-well plate, then treated with postotic osdine and incubated for 24 hours. The control group was treated with the same amount of DMSO without adding posture osdine. After the treatment time, a solution of MTT (3- (4,5) -dimethylthiahiazo (-z-y1) -3,5-di-phenytetrazoliumromide) was added to each well and reacted for 3 hours. Absorbance was measured at.

As a result, as shown in FIG. 1A, postural osdine did not significantly affect the viability of 3T3-L1 adipocytes at a concentration of 0-80 μM.

Experimental Example 2. Confirmation of adipose cell differentiation inhibitory effect of postural oscidin

3T3-L1 adipocytes cultured in Example 1 were observed through a microscope at 400x magnification, and are shown in FIG. 1B. When 3T3-L1 adipocytes cultured with postural osdine were compared with the negative control (NC) using adipocytes and the positive control (PC) using fully differentiated adipocytes, the concentration of 3T3-L1 adipocytes was 3T3. -L1 adipocyte differentiation was strongly inhibited.

Experimental Example 3. Confirmation of the effect of postural osdine on cell lipid accumulation

Lipid staining was performed using Oil Red O (ORO) staining technique and triglyceride content was performed according to the manufacturer's manual using TG Quantification Kit (Bio Vision). For lipid staining, 3T3-L1 adipocytes cultured in Example 1 were fixed with 0.6% glutaraldehyde, stained with 0.4% ORO solution, and washed three times with distilled water. Stained lipids were observed through a microscope at 200x magnification, and dissolved ORO reagent using 100% isopropyl alcohol, and then absorbance at 495 nm was measured and quantified.

As a result, as shown in FIG. 2A, lipid accumulation in 3T3-L1 adipocytes was normally induced in the positive control group, but lipid accumulation was reduced in a concentration-dependent manner in 3T3-L1 adipocytes treated with postiodine.

In addition, to measure the content of triglycerides, 3T3-L1 adipocytes cultured in Example 1 were dissolved in a lysate containing 4% NP-40, centrifuged to take a supernatant, and according to the manufacturer's manual. Intracellular TG content was analyzed by measuring absorbance at 570 nm.

As a result, as shown in FIG. 2B, the TG content in 3T3-L1 adipocytes treated with 10, 20, and 40 μM of postiosine was significantly decreased in comparison with the positive control group at 87.4, 81.3, and 72.8%, respectively.

Therefore, it can be seen from the above results that postiosidine reduces intracellular lipid accumulation during the differentiation of 3T3-L1 adipocytes into mature adipocytes.

Experimental Example  4. Ossidine  Transcription factors related to adipocyte formation and differentiation, and Adiponectin  Confirmation of expression inhibition effect

4-1. mRNA  Expression analysis

Cell lysis using total RNA extract reagents (RNAiso Plus, TAKARA, Japan) from 3T3-L1 adipocytes treated in Example 1, chloroform was added to the supernatant by centrifuge for 15 minutes at 4 ℃, 12000rpm Got. The supernatant was mixed with isopropanol, reacted, centrifuge for 10 minutes under the same conditions, precipitated RNA, and washed with 75% ethanol to separate RNA. The extracted RNA was synthesized into cDNA using PrimeScript 1st strand cDNA Synthesis Kit (TAKARA).

Thereafter, real-time PCR (RT-PCR) was performed using the synthesized cDNA. RT-PCR is Real-time PCR Detection System (Bio-Rad Laboratories, USA) equipment was used to measure and compare the luminescence level of SYBR Green. Primer type and sequence of the detected genes are shown in Table 1.

As a result, as shown in FIG. 3A, it was found that the postureodine inhibits mRNA expression of PPARγ, C / EBPa, and adiponectin in a concentration-dependent manner.

4-2. Protein expression analysis

3T3-L1 cells treated in Example 1 were lysed using lysis buffer and centrifuged to obtain supernatant. Then, total protein was quantified by Bradford method and electrophoresed with the same amount of protein (30μg) in 10% SDS polyacrylamide gel. After electrophoresis, the protein in SDS polyacrylamide gel was transferred to Nitrocellulose Blotting Membrane (Amersham Protran Premium 0.2μm NC, GE Healthcare Life Sciences, Germany). 5% skim milk was applied to the protein-transferred membrane to block nonspecific proteins for 1 hour. After washing the membrane, the primary antibody was reacted with a secondary antibody appropriate for the treated primary antibody. After completion of the reaction, it was detected by reacting with Enhanced Chemiluminoesence (ECL) solution.

As a result, as shown in FIG. 3B, it was confirmed that vesicular oscillin inhibits PPARγ2 and adiponectin protein expression.

Example 2 Statistical Analysis

All experiments were repeated three times, and one-way batch analysis was used for statistical analysis. p <0.05 indicates statistical significance.

The specific parts of the present invention have been described in detail above, and it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. something to do. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (10)

Agobiodine containing as an active ingredient, obesity prevention or treatment pharmaceutical composition.
The method of claim 1,
The composition is characterized in that to inhibit the differentiation of fat cells, obesity prevention or treatment pharmaceutical composition.
The method of claim 1,
The composition is characterized in that to reduce the accumulation of lipids in the cell, obesity prevention or treatment pharmaceutical composition.
The method of claim 1,
The composition is characterized in that to suppress the expression of a transcription factor or adiponectin involved in adipocyte formation and differentiation, obesity prevention or treatment pharmaceutical composition.
The method of claim 4, wherein
The transcription factor is characterized in that the peroxysomal proliferator activator receptor gamma (PPARγ) or CCAAT-enhancer binding protein alpha (C / EBPa), a pharmaceutical composition for preventing or treating obesity.
Agotiodine containing as an active ingredient, obesity prevention or improvement health functional food composition.
The method of claim 6,
The composition is characterized in that to inhibit the differentiation of fat cells, obesity prevention or improvement health functional food composition.
The method of claim 6,
The composition is characterized in that to reduce the accumulation of lipids in the cell, health food composition for preventing or improving obesity.
The method of claim 6,
The composition is characterized in that to suppress the expression of transcription factors or adiponectin involved in adipocyte formation and differentiation, obesity prevention or improvement health functional food composition.
The method of claim 9,
The transcription factor is characterized in that the peroxysomal proliferator activator receptor gamma (PPARγ) or CCAAT-enhancer binding protein alpha (C / EBPa), a health functional food composition for preventing or improving obesity.

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