KR20190125068A - A composition for preventing or improving obesity or obesity-related disease comprising 6'-O-acetyl mangiferin - Google Patents

Abstract

The present invention relates to a composition for preventing or alleviating obesity or obesity-related diseases comprising 6′-O-acetyl mangiferin (OAM) as an active component. OAM according to the present invention down-regulates mRNA expression and gene expression of adipocyte differentiation transcription factors and proteins, and controls lipolytic enzymes and ACC through activation of an AMPK pathway to up-regulates lipolysis and β-oxidation, thereby significantly reducing a triglyceride level in cells. Therefore, it is possible to be usefully used as the active component in pharmaceutical and food compositions for preventing or treating obesity or obesity-related diseases.

Description

A composition for preventing or improving obesity or obesity-related disease comprising 6'-O-acetyl mangiferin}

The present invention relates to a composition for the prevention or improvement of obesity or obesity-related diseases comprising 6'-O-acetyl mangiferin (OAM) as an active ingredient, more specifically AMP-activity A pharmaceutical or food composition for the prevention or improvement of obesity or obesity-related diseases comprising 6'-O-acetyl manjiferin having an inhibitory effect on lipid production accumulation by activated protein kinase (AMPK) activity will be.

As mankind has developed into a prosperous society, obesity has emerged as one of the serious diseases, and the World Health Organization (WHO) has declared it the object of the disease to be treated. Obesity is a metabolic disease caused by an imbalance between calorie intake and consumption and is morphologically caused by hypertrophy or hyperplasia of fat cells in the body. Obesity is not only the most common malnutrition in Western society, but the importance of treatment and prevention has been highlighted in recent years in Korea, as the frequency of obesity is rapidly increasing due to the improvement of dietary life and the westernization of lifestyle. have. Obesity is an important factor that not only psychologically diminishes individuals but also increases the risk of developing various adult diseases. Obesity is known to be directly related to increased prevalence of various adult diseases, such as type 2 diabetes, hypertension, hyperlipidemia, and heart disease (Cell 87: 377, 1999), and the combination of obesity-related diseases together with metabolic syndrome or insulin resistance It is called syndrome (insulin resistance syndrome), and these have been found to be the cause of atherosclerosis and cardiovascular disease. As such, obesity increases the incidence of various metabolic diseases, and the fact that weight loss significantly reduces the incidence of these diseases can be inferred that fat-rich fat cells mediate this phenomenon.

In the past, adipose tissue was only thought of as an energy storage organ that stores excess energy in the form of triglyceryl (triacylglycerol) and releases it when needed, but in recent years various adidies such as adiponectin, leptin and resistin Adipokines are accepted as important endocrine organs that regulate homeostasis of energy (Trends Endocrinol Metab 13:18, 2002). Therefore, understanding of the proliferation of fat cells and the substances secreted from fat cells and their in vivo regulation mechanisms are expected to be the foundation for understanding obesity and various diseases and developing effective treatments. Studies on the regulation of adipocyte differentiation are being actively conducted, and the main mechanism is that differentiation from preadipocytes in the body is associated with increased adipocyte derivation in obese patients.

The process of differentiation of pro-adipocytes into adipocytes has been studied using cells such as 3T3-L1, and several transcription factors, especially transcription factors known to be involved in localization, C / EBPs (CCAAT enhancers). It is known that binding proteins, PPARs (Peroxisome proliferator activated receptor) and ADD1 / SREBPs (Adipocyte determination and differentiation dependent factor1 / sterol response element binding proteins) are expressed over time and regulate the process (Bart A Jessen). et al., Gene, 299, pp95-100, 2002; Darlington et al., J. Biol. Chem., 273, pp30057-30060, 1998; Brun RP et al., Curr. Opin. Cell. Biol., 8 , pp826-832, 1996). Given the stimulation of hormones such as isobutylmethylxanthin, dexamethasone and insulin (MDI), C / EBP β and δ are the first, transiently expressed, to initiate differentiation into adipocytes (Reusch J. E et al., Mol Cell Biol., 20, pp 1008-1020, 2000). This continues to lead to increased expression of C / EBPa and PPARγ (James M. N. et al., J. Nutr., 130, pp 3122S-3126S, 2000). PPARγ is a particularly important transcription factor for adipocyte differentiation, and forms dimers with the retinoic acid X receptor protein (RXR), which is present in the promoters of various adipocyte genes. Binds to peroxisome proliferator response elements (Tontonoz PE et al., Genes Dev., 8, pp1224-1234, 1994; Hwang, C. S et al., Cell Dev. Biol., 13, pp873-877) . The interaction of PPARγ with C / EBP α is critical for differentiation into mature adipocytes. These transcription factors and adipocyte modulators promote the differentiation of adipocytes into adipocytes, and with ap2 (adipocyte fatty acid binding protein 2) Expression levels of fat cell specific proteins such as fat fat enzymes such as fat acid synthase (FAS) are increased. In addition, ADD1 / SREBPs play an important role in fat metabolism, but are also known to be involved in the differentiation process. Expression of ADD1 / SREBP1c in immature adipocytes is believed to contribute to the activation of PPARγ (Rosen ED et al., Annu. Rev. Cell Dev. Biol., 16, pp 145-171, 2000; Osborn TF, J. Biol Chem., 275, pp 32379-32382, 2000). Only the differentiated fat cells synthesize fatty acids and store triglycerides. Therefore, current research trends are focused on the discovery of substances that can inhibit metabolic processes related to adipocyte differentiation as a method for preventing or treating obesity and lipid-related metabolic diseases. In other words, attempts have been made to treat obesity through the regulation of fat cells based on the mechanism of the development of obesity, which aims to reduce fat synthesis by inhibiting fat synthesis or promoting fat breakdown and oxidation, and inhibiting fat cell differentiation. To reduce cell numbers, transcription factors, proteins and adipokines, known to mediate or regulate these processes, have emerged as targets for the development of new anti-obesity drugs. Indeed, the PPAR family of adipocyte differentiation transcription factors, leptin and adiponectin, adipocyte secreting substances, have been targeted for many new drug developments.

Known obesity treatments such as Xenical (Roche Pharmaceuticals, Switzerland), Reductil (Eboth, USA), Exolise (Atopama, France), etc. Classified as inhibitors, most anti-obesity agents are appetite suppressants that suppress appetite by regulating neurotransmitters associated with the hypothalamus. However, the conventional treatments have low side effects such as heart disease, respiratory disease, and neurological disease, and thus have low continuity. Therefore, the development of improved obesity drugs is needed, and the currently developed products have satisfactory treatment effects without side effects. Since there are few therapeutic agents, development of new obesity agents is required.

On the other hand, Mangiferin, a plant chemical, has various physiological activities including neuroprotection, cardioprotection, gastric protective function, anti-obesity, anti-inflammatory and antidiabetic effects (M. Sekar, Molecules of Interest-Mangiferin, Annu) Res Rev Biol. 5 (2015) 307-320). Manziferin has been reported to regulate plasma lipid levels (reduce total cholesterol, triglycerides, free fatty acids (FFAs), increase high-density lipoprotein cholesterol) in diabetic animal models (F. Guo, et al., Mol. Nutr. Food). Res. 55 (2011) 1809-1818 and F. Cai, et al., J. Chromatogr.B. Analyt Technol.Biomed.Life.Sci. 878 (2010) 1845-1854]. Recently, it has been reported that manjiferin inhibits cell differentiation by attenuating the expression of fat forming genes including fatty acid binding protein 4 (FABP4) and PPARγ in human mesenchymal stem cells (hMSCs) (J. Han, Mol. Cell. Endocrinol. 405 (2015) 63-73). X-3, a new derivative of manjiferin, mediates the activation of AMPK and AKT and has recently emerged as a new antidiabetic drug. Manziferin Derivative 6'-O-acetyl Manjiferrin (OAM) has been reported to inhibit nitric oxide, prostaglandin E2 and pro-inflammatory cytokines by inhibiting NF-κB and MAPK signaling pathways, but OAM has an anti-obesity effect The fact that there is not yet known.

Accordingly, the technical problem to be solved in the present invention is to provide a novel substance having the effect of preventing or treating obesity or obesity-related diseases.

In order to solve the above technical problem, in the present invention 6'-O-acetyl mangiferin (6'-O-acetyl mangiferin) or a pharmaceutically acceptable salt thereof as an active ingredient prevention of obesity or obesity-related diseases Or it provides a pharmaceutical composition for treatment.

According to one embodiment of the present invention, in the present invention, 6'-O-acetyl mangiferin (6'-O-acetyl mangiferin) or a food-acceptable salt thereof as an active ingredient of obesity or obesity-related diseases It provides a food composition for prevention or improvement.

6'-O-acetyl mangiferin as an active ingredient of the composition of the present invention is a compound having the formula (1) can be isolated from each iris or chemically synthesized.

In the present invention, when the 6'-O-acetyl manjiferin is separated from each iris, it can be extracted using an extraction solvent commonly used in the art, the extraction solvent is, for example, (a) water (b) anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, propanol, butanol, etc.), (c) a mixed solvent of the lower alcohol with water, (d) acetone, (e) ethyl acetate, ( f) chloroform or (g) 1,3-butylene glycol can be used. On the other hand, it will be apparent to those skilled in the art that the 6'-O-acetyl manjiferin of the present invention can be obtained in addition to the above-described extraction solvents, and extracts having substantially the same effect using other extraction solvents.

In addition, the 6'-O-acetyl manjiferin can be obtained through a conventional separation and purification in addition to the extraction method by the extraction solvent. 6'-O-acetyl manziferin is obtained through fractions obtained through various purification methods additionally performed, for example, separation by various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity). can do.

According to one embodiment of the present invention, the present invention relates to a novel use of 6'-O-acetyl manjiferin, wherein 6'-O-acetyl manjiferin or a pharmaceutically or pharmaceutically acceptable salt thereof is an active ingredient. It provides a composition for the prevention, improvement or treatment of obesity or obesity-related diseases comprising.

As used herein, the terms "pharmaceutically acceptable" and "food acceptable" mean that they do not stimulate the organism and do not inhibit the biological activity and properties of the administered active substance.

6'-O-acetyl manjiferin or a pharmaceutically or food-acceptable salt thereof according to the present invention can be used as an active ingredient for preventing, ameliorating or treating obesity or obesity-related diseases. The obesity-related diseases are not limited in kind as long as they are caused by obesity or are highly correlated with obesity, for example, selected from fatty liver, type 2 diabetes, hyperlipidemia, cardiovascular disease, arteriosclerosis, and lipid-related metabolic syndrome. It can be any one. In addition, the lipid-related metabolic syndrome refers to a disease in which various metabolic diseases, such as diabetes and obesity, appear simultaneously in one person.

The composition comprising the 6'-O-acetyl manjiferin of the present invention or a pharmaceutically or food-acceptable salt thereof as an active ingredient is a pharmaceutical composition, a food additive, a food composition (particularly a functional food composition) according to the purpose or aspect of use. It may be embodied as a feed additive, or the like, and the content of the active ingredient in the composition may be adjusted in various ranges according to the specific form of the composition, the purpose of use, and the aspect thereof.

In the pharmaceutical composition comprising 6'-O-acetyl manjiferin or a pharmaceutically acceptable salt thereof according to the present invention as an active ingredient, the content of the active ingredient is not particularly limited, for example, 0.001 based on the total weight of the composition. To 50% by weight, preferably 0.01 to 30% by weight, more preferably 0.01 to 10% by weight. When the content of the 6'-O-acetyl manjiferin or a pharmaceutically acceptable salt thereof is less than 0.001% by weight, the effect of inhibiting fat decomposition and prevention is weak, and when the content exceeds 50% by weight, the increase in effect is increased. It may be inefficient because it is not an enemy, there is a problem that the stability of the formulation is not secured.

In addition, the pharmaceutical composition according to the present invention may further include additives such as pharmaceutically acceptable carriers, excipients, or diluents, in addition to 6'-0-acetyl manjiferrin or a pharmaceutically acceptable salt thereof. Carriers, excipients and diluents that may be included in the pharmaceutical compositions of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate and mineral oil.

In addition, the pharmaceutical composition for the prevention or treatment of obesity or obesity-related diseases of the present invention is a known active ingredient having a prophylactic or therapeutic effect of obesity or obesity-related diseases in addition to the pharmaceutically acceptable salt of 6'-O-acetyl manjiferin It can contain 1 or more types.

6'-O-acetyl manjiferin (OMA) of the present invention not only has a very low accumulation of lipids and triglycerides in the cell but also decreases lipid production genes and proteins to inhibit fat production. 3T3-L1 cells treated with OAM excreted more glycerol, indicating increased lipolysis, which would be confirmed by increased expression of lipolysis related genes, including lipid triglyceride lipase and hormone-sensitive lipase. Can be. In addition, OAM upregulated the β-oxidation pathway with increased expression of phosphorylated acetyl-CoA-carboxylase and long chain acyl-CoA synthase 1.

In accordance with specific embodiments of the present invention, the effects of adipocyte differentiation related protein expression and mRNA expression were investigated to understand the molecular mechanism of OAM.

Adipocyte differentiation is a multistep process involving a complex network of transcription factors. Hormonal stimulation increases the expression of the transcription factors C / EBPβ and C / EBPδ early in differentiation, and these transcription factors in turn induce the expression of PPARγ and C / EBPα (DP Ramji, P. Foka, Biochem. J. 365). (2002) 561-575 and QQ Tang, et al., Proc. Natl. Acad. Sci. USA 102 (2005) 9766-9771). PPARγ and C EBPα are expressed at early and intermediate stages, respectively. Later these transcription factors regulate the expression of fat cell specific genes, including FABP4 and FASN, to form lipid droplets (J.W. Yang, et al., Molecules. 20 (2015) 1293-1303). SREBP-1c is one of the fat production-related transcription factors that regulates late stages of adipocyte differentiation (S. Chen, et al, Can. J. Physiol. Pharmacol. 89 (2011) 793-799). Thus, inhibition of adipose development-related transcriptional expression is considered a useful method for treating obesity.

According to a specific embodiment of the present invention, OAM downregulates mRNA and protein expression of PPARγ, C / EBPa, FASN and FABP4 and mRNA expression of SREBP-1c, adipose-related transcription and their down-regulation enzymes Lipid accumulation was inhibited in the cells.

Imbalance of lipid metabolism (between fatty acid β-oxidation and synthesis) leads to excessive lipid accumulation, which is associated with various metabolic disorders. Fatty acid molecules are metabolized through β oxidation, and upregulation of this reaction reduces the fatty acids in the cells that can be used for triglyceride synthesis. As a result, β-oxidation can reduce triglyceride production. In addition, ACC blocks malonyl-CoA synthesis to reduce triacyl glycerol accumulation and increase fatty acid oxidation (H. Asano, et al., J. Vet. Med. Sci. 76 (2014) 57-64). Acsl1 plays an important role in mitochondrial and peroxysome beta oxidation through the conversion of long chain FFAs into intracellular fat acyl-CoA (M.O. Sim, et al., J. Funct. Food. 15 (2015) 160-171).

According to a specific embodiment of the present invention, OAM stimulated mRNA and protein expression of p-ACC and Acsl1 such that OAM increased β-oxidation, thereby reducing triglyceride production.

According to a specific embodiment of the present invention, glycerol release was investigated to investigate whether inhibition of lipid accumulation by OAM is associated with lipolysis.

Lipolysis is the process of triglyceride (TG) and release of free fatty acids (FFA) and glycerol, and the pathway is initiated by two major enzymes, ATGL and HSL (BR Sharma, et al., J. Integr. Med). 15 (2017) 56-63). First, ATGL promotes triglyceride hydrolysis to produce FFA and diacylglycerols that are degraded by HSL.

According to a specific embodiment of the invention, 3T3-L1 adipocytes in the presence of OAM increased glycerol release levels, increased mRNA expression of ATGL and HSL, and increased protein expression of p-HSL and ATGL. OAM significantly reduced intracellular triglyceride levels by promoting lipolysis through ATGL and HSL and up-regulation of β-oxidation through p-ACC and Acsl1.

Since AMPK is a molecular candidate for regulating fat production and β-oxidation of adipose tissue, AMPK is emerging as an attractive target for the treatment of metabolic disorders by regulating a wide range of cellular metabolic pathways including lipid homeostasis (HJ Ko, et al. , J. Funct. Food. 17 (2015) 271-282). It has been reported that the AMPK pathway can inhibit adipocyte differentiation by blocking the transcription of adipogenic genes including C / EBPa and PPARγ (BC Cheng, et al., J. Ethnopharmacol. 153 (2014) 922-927 And B. Gao, R. Bataller, et al., Gastroenterology. 141 (2011) 1572-1585]. SREBP1 and FAS are downstream genes of AMPK. In addition, there are various studies in which AMPK inhibits the phosphorylation-mediated activation of ACC (Y. He, et al., PLoS One. 8 (2013) e70135). ACC is an enzyme that limits the rate important in the oxidation and synthesis of fatty acids and contributes to the progression of obesity. AMPK is expected to reduce lipid synthesis and increase fatty acid oxidation because it inhibits the activation of ACC and adipose development-related transcription factors (M.H. Han, et al., Toxicol. Res. 34 (2018) 13-21). In addition, AMPK has been reported to upregulate two lipolytic regulatory enzymes, ATGL and HSL, in adipose tissue (S.J. Kim, et al., Mol. Cell. Biol. 36 (2016) 1961-1976).

According to a specific embodiment of the present invention, OAM treatment increases the phosphorylation of AMPK and inhibits ACC, PPARγ, C / EBPa and SREBP-1 such that AMPK phosphorylation is responsible for the reduction of adipocyte differentiation and upregulation of β oxidation by OAM. It was confirmed that it was involved. In addition, OAM treatment also increased ATGL and p-HSL. These results suggest that OAM upregulates lipolysis through activation of AMPK.

Thus, according to one embodiment of the invention, relates to a novel use of 6'-O-acetyl manziferin, obesity or obesity associated with genthiopicroside or a pharmaceutically acceptable salt thereof as an active ingredient It provides a pharmaceutical composition for the prevention or treatment of diseases.

As used herein, the term "prevention" refers to any action that inhibits the symptoms or delays the progression of a particular disease, such as obesity or obesity-related disease, by administration of a composition of the invention.

As used herein, the term "treatment" refers to any action that improves or beneficially alters the symptoms of a particular disease, such as obesity or an obesity related disease, by administration of a composition of the present invention.

The pharmaceutical composition is any one selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, liquid solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilizers and suppositories. It can have a formulation of, and can be a variety of oral or parenteral formulations. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which form at least one excipient such as starch, calcium carbonate, sucrose or lactose (at least one compound). lactose) and gelatin. In addition to simple excipients, lubricants such as magnesium stearate, talc and the like are also used. Liquid preparations for oral administration include suspensions, liquid solutions, 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 sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The composition of the present invention may be administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" refers to 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 and severity, age, sex, type of disease of the individual. , The activity of the drug, 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 concurrent drug and other factors well known in the medical field. The compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents. And single or multiple administrations. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, and can be easily determined by those skilled in the art. The preferred dosage of the composition of the present invention depends on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration of time, and the suitable total daily dosage can be determined by the practitioner within the correct medical judgment. Generally, an amount of 0.001 to 1000 mg / kg, preferably 0.05 to 200 mg / kg, more preferably 0.1 to 100 mg / kg, may be administered once to several times daily. The composition is not particularly limited as long as it is a subject for anti-obesity purpose, and any subject may be applied. For example, it can be applied to any individual such as non-human animals such as monkeys, dogs, cats, rabbits, marmots, rats, mice, cows, sheep, pigs, goats, humans, birds, and fish, and the mode of administration is in the art. Any conventional method may be used without limitation. For example, it may be administered by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injections.

In one specific embodiment of the present invention, it provides a food composition for the prevention or improvement of obesity or obesity-related diseases, containing 6'-O-acetyl manjiferin as an active ingredient.

In the present invention, the term "improvement" refers to all the actions that improve or benefit the symptoms of suspected and onset individuals with obesity using the composition.

When using the composition of the present invention in food, the composition may be added as it is, or used with other health functional foods or health functional food ingredients, and may be appropriately used according to conventional methods. The mixing amount of the active ingredient may be appropriately determined depending on the intended use. In general, the composition of the present invention may be added in the amount of preferably 15 parts by weight or less, more preferably 10 parts by weight or less based on the raw material in the manufacture of food or beverage. However, in the case of long-term intake for the purpose of health control and hygiene, the amount may be below the above range, and since there is no problem in terms of stability, the active ingredient may be used in an amount above the above range.

There is no particular limitation on the kind of foods that may include the composition of the present invention, and specific examples include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, and ice cream. , Various soups, beverages, tea, drinks, alcoholic beverages and vitamin complexes, etc., may include all of the food in the usual sense, and may include foods used as feed for animals. In addition, when the food composition of the present invention is used in the form of a beverage, various sweeteners, flavoring agents or natural carbohydrates, etc. may be contained as additional components, as in general beverages. The natural carbohydrate may be glucose, monosaccharides such as fructose, maltose, disaccharides such as sucrose, polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, erythritol. The ratio of the natural carbohydrate is not limited thereto, but may be preferably about 0.01 to 0.04 g, more preferably 0.02 to 0.03 g per 100 ml of the composition of the present invention. The sweetener may be a natural sweetener such as taumartin, stevia extract and a synthetic sweetener such as saccharin, aspartame. In addition to the above, the food composition of the present invention includes various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols. And carbonation agents used in carbonated beverages. Others may contain pulp for the production of natural fruit juices, fruit juice drinks and vegetable drinks.

In another aspect, the present invention provides an anti-obesity method comprising administering the pharmaceutical composition to a suspicious subject of obesity. In the present invention, the suspicious subject of obesity refers to all animals including humans who may or may develop obesity, and the pharmaceutical composition comprising a compound of the present invention or a pharmaceutically acceptable salt thereof may be added to a suspicious subject of obesity. By administering, the individual can be treated efficiently.

As used herein, the term "administration" refers to the introduction of the pharmaceutical composition of the present invention to a subject of suspected obesity by any suitable method, and the route of administration may be administered via various routes orally or parenterally as long as the target tissue can be reached. Can be. The method of treatment of the present invention may comprise administering a pharmaceutical composition comprising the 6'-O-acetyl manjiferin in a pharmaceutically effective amount. Suitable total daily doses may be determined by the practitioner within the correct medical judgment and are generally in amounts of 0.001 to 1000 mg / kg, preferably 0.05 to 200 mg / kg, more preferably 0.1 to 100 mg / The amount of kg may be administered once to several times daily.

However, for the purposes of the present invention, the specific therapeutically effective amount for a particular patient is determined by the specific composition, including the type and extent of the reaction to be achieved, whether or not other agents are used in some cases, the age, weight, general health of the patient, It is desirable to apply differently depending on various factors and similar factors well known in the medical field, including sex and diet, time of administration, route of administration and rate of composition, duration of treatment, drugs used with or co-specific with the specific composition.

On the other hand, 6'-O-acetyl manjiferin of the present invention is a natural substance, harmless to the human body, there is almost no toxicity and side effects, so it can be used safely even in the long-term use, especially safely applied to the pharmaceutical and food compositions as described above can do.

In one specific embodiment, the present invention provides a method and use thereof for preventing or improving obesity in an individual by administering to the individual an anti-obesity composition comprising 6'-O-acetyl manjiferin as an active ingredient.

As described above, according to the present invention, 6'-O-acetyl manjiferin down-regulates mRNA expression and gene expression of adipocyte differentiation transcription factors and proteins, and regulates lipolytic enzymes and ACC through activation of the AMPK pathway. And up-regulating β-oxidation to significantly reduce intracellular triglyceride levels, which can be safely used as an active ingredient in pharmaceutical and food compositions for the prevention or treatment of obesity or obesity-related diseases.

1 shows the effect of OAM on lipid droplet accumulation in differentiated 3T3-L1.
2 shows the effect of OAM on lipid synthesis in differentiated 3T3-L1.
3 shows the effect of OAM on lipid degradation in differentiated 3T3-L1.
4 shows the effect of OAM on β-oxidation in differentiated 3T3-L1.
5 shows the effect of OAM on the expression of AMPK in differentiated 3T3-L1.

Hereinafter, examples and the like will be described in detail to help understand the present invention. However, embodiments according to the present invention can be modified in many different forms, the scope of the invention should not be construed as limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

<Example 1>

1. Experimental method

1-1. Cell Culture and Differentiation

3T3-L1 (pre-adipocyte) cells were purchased from ATCC. To induce adipocyte differentiation, cells (1 x 10 ⁵ cells / well) were plated in 60 mm cell culture dishes and culture medium (DMEM containing 1% PS, 10% BCS) was replaced every 2 days. After 4 days, when all cells reached confluence (indicated on day 0), the medium was replaced with MDI differentiation medium (1% P / S, 10% FBS, 10 μg / mL insulin, 1 μg DEX and 0.5 mM IBMX). Replaced with. After 2 days (day 2), cells were maintained in medium containing only insulin for 2 days. Thereafter (day 4) medium was replaced with post-differentiation medium (DMEM containing 1% P / S, 10% FBS) for another 4 days and then every 2 days. Different concentrations of 6′-O-acetyl manziferin (OAM, 25, 50, 100 μM) were added to the medium for 0-8 days and the controls were treated in equal volumes and DMSO concentrations were used as controls.

1-2. Determination of Cell Viability and Lipid Accumulation

The effect of OAM on cell viability in 3T3-L1 cells was measured using a commercial kit of MTS assay. Lipid accumulation was determined by Oil Red O staining using a commercial kit (ScienCell, Carlsbad, Calif., USA) and measuring triglyceride content 8 days after induction of differentiation. Differentiated cells were fixed in fixed solution for 1 hour at room temperature and washed with PBS. After complete drying, the cells were stained with Oil Red O solution for 30 minutes at room temperature and washed four times with distilled water. Stained cells were observed at 200x magnification under a microscope (Nikon E100 microscope; Nikon Instruments Europe, Kingston upon Thames, UK). To quantify staining, 100% isopropanol was added and the plate was slowly rotated for 10 minutes and absorbance was read using an Infinite 200 Pro instrument. To analyze intracellular triglyceride levels, 3T3-L1 cells were harvested and lysed in RIPA cell lysis buffer. Supernatants were used to analyze triglyceride levels using a commercial kit (Asan Pharmaceutical, Seoul, Korea).

1-3. Determination of Lipolysis by Glycerol Release Analysis

3T3-L1 cells were differentiated for 8 days and culture medium was collected. Medium was used to determine the glycerol released into the medium using the Glycerol Quantitation Kit (BioVision, Milpitas, Calif., USA) according to the manufacturer's instructions.

1-4. Lipidogenesis-related mRNA expression

Lipidogenesis-related mRNA expression is described in the previously described methods [M.O. Sim, et al., Biomed. Pharmacother. 93 (2017) 165-171] was used.

1-5. Western Blotting

Adipocytes were collected and lysed in RIPA cell lysis buffer. The lysates were then denatured at 95 ° C. for western blotting analysis. Protein (30 μg) was separated on 7-12% SDS-polyacrylamide gel (Bio-Rad, Richmond, CA, USA) and then transferred to PVDF membrane (GE Healthcare, Dassel, Germany). After blotting in 5% bovine serum albumin in Tris-HCl buffer, the membrane was AMPK, p-AMPK, fatty triglyceride lipase (ATGL), hormone sensitive lipase (HSL), p-HSL, p-HSL, PPARγ, C / EBPα, acetyl-CoA-carboxylase (ACC), p-ACC, mammalian long chain acyl-CoA synthase 1 (Acsl1), incubated overnight at 4 ° C with antibodies to FABP4 and GAPDH. The membrane was then incubated with the secondary antibody for 2 hours. Protein bands were then visualized using ECL reagents and imaged using an imaging system (LiCor / Odyssey, LI-COR Biosciences, Lincoln, NE, USA).

1-6. Statistical analysis

All data are expressed as mean W standard deviation (SD). Statistical analysis was performed using the Statistical Package for the Social Science, Ver. 12.0 SPSS Inc., Chicago, IL, USA. Differences in mean values of the OAM treatment groups were confirmed by Duncan's multiple range test using ANOVA analysis. p <0.05 was considered statistical significance.

2. Results

2-1. Effect of OAM on Lipogenesis and Intracellular Triglyceride Levels

To investigate the potential inhibitory effects of OAM on lipid formation, investigate the effect of OAM on lipid accumulation and intracellular triglyceride levels in all concentrations of 3T3-L1 cells that do not affect cell viability as determined by MTS analysis. It was. After differentiation, cells were stained with Oil Red O to quantify intracellular lipid accumulation.

1 shows the effect of OAM on lipid droplet accumulation in differentiated 3T3-L1. As shown here, lipid droplets were seen in fully differentiated control cells grown in MDI treated groups, but OAM treatment reduced the number of lipid droplets in a dose dependent manner (FIG. 1A). This was confirmed by quantification of oil red O staining (FIG. 1B). In addition, OAM treatment reduced intracellular triglyceride levels by about 52% at 50 μΜ relative to control cells (FIG. 1C). These results thus suggest that OAM inhibits lipid formation and triglyceride accumulation.

2-2. Effect of OAM on the Expression of Lipid Genes and Proteins

We investigated the effects of OAM on the formation of transcription factors and lipid producing genes and proteins associated with lipid production.

2 shows the effect of OAM on lipid synthesis in differentiated 3T3-L1. As shown here, OAM downregulated mRNA and protein expression of C / EBPa and PPARγ as well as mRNA expression of SREBP-1c. FABP4 binds fatty acids and is transferred to various cell compartments. Fasn is an important enzyme in fatty acid synthesis pathways. Fasn and FABP4 expression was significantly decreased in OAM treatment compared to control.

2-3. Effect of OAM on Lipolysis

To assess the effect of OAM on lipolysis, the release of glycerol by 3T3-L1 cells was measured upon treatment. 3 shows the effect of OAM on lipid degradation in differentiated 3T3-L1. As seen here, OAM significantly increased glycerol release at all doses. In addition, OAM treatment upregulated p-HSL and ATGL protein levels as well as mRNA expression of HSL and ATGL.

2-4. Effect of OAM on β-oxidation

To elucidate the mechanism by which OAM reduces intracellular triglyceride levels, we evaluated gene and protein expression associated with fatty acid β-oxidation pathways. 4 shows the effect of OAM on β-oxidation in differentiated 3T3-L1. As shown here, both phosphorylation of ACC and mRNA expression of ACC were increased by OAM treatment. OAM treatment also increased the mRNA and protein expression of Acs1.

2-5. Effect of OMP on the Expression of AMPK

AMPK is an important target for treating metabolic diseases through the regulation of lipid biosynthetic pathways. 5 shows the effect of OAM on the expression of AMPK in differentiated 3T3-L1. As shown here, OAM increased the expression of p-AMPK in adipocytes after dose-dependent treatment with OAM.

Claims (4)

Pharmaceutical composition for the prevention or treatment of obesity or obesity-related diseases comprising 6'-O-acetyl manjiferin or a pharmaceutically acceptable salt thereof as an active ingredient. The method of claim 1,
The obesity-related diseases are fatty liver, type 2 diabetes, hyperlipidemia, cardiovascular disease, atherosclerosis and lipid-related metabolic syndrome, characterized in that the pharmaceutical composition for the prevention or treatment of obesity or obesity-related diseases. Food composition for the prevention or improvement of obesity or obesity-related diseases comprising 6'-O-acetyl manjiferin or a food acceptable salt thereof as an active ingredient. The method of claim 3, wherein
Food composition for the prevention or improvement of obesity or obesity-related diseases, characterized in that the obesity-related disease is selected from the group consisting of fatty liver, type 2 diabetes, hyperlipidemia, cardiovascular disease, arteriosclerosis and lipid-related metabolic syndrome.

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