KR20170129164A - A composition for inhibiting proliferation of adipose stem cells and for promoting browning of adipose tissue - Google Patents

Abstract

The present invention relates to an adipocyte stem cell or a lipogenic precursor cell comprising at least one selected from the group consisting of mangiferin, neomangiferin, norathriol, and pharmaceutically acceptable salts thereof. A composition for inhibiting proliferation or differentiation of the cell; A composition for promoting the differentiation of adipose tissue into brown adipocytes or beige adipocytes; A composition for inhibiting the differentiation of adipose tissue into white adipocytes; A composition for preventing, ameliorating, or treating hypothermia; Compositions for controlling body temperature or regulating body energy metabolism; A composition for preventing, ameliorating or treating a lipid metabolism-related disease; And anti-obesity, or compositions for reducing body fat.

Description

A composition for inhibiting proliferation of adipose stem cells and for promoting browning of adipose tissue

The present invention relates to an adipocyte stem cell or a lipogenic precursor cell comprising at least one selected from the group consisting of mangiferin, neomangiferin, norathriol, and pharmaceutically acceptable salts thereof. A composition for inhibiting proliferation or differentiation of the cell; A composition for promoting the differentiation of adipose tissue into brown adipocytes or beige adipocytes; A composition for inhibiting the differentiation of adipose tissue into white adipocytes; A composition for preventing, ameliorating, or treating hypothermia; Compositions for controlling body temperature or regulating body energy metabolism; A composition for preventing, ameliorating or treating a lipid metabolism-related disease; And compositions for anti-obesity, appetite suppression, or body fat reduction.

Obesity is basically caused by excessive accumulation of surplus energy in the form of fat in the body when the energy consumed is smaller than the energy consumed. In the modern society, the intake of energy by the westernization of the diet is greatly increased, but the disparity between the ingestion energy and the consumed energy easily occurs by decreasing the activity amount through convenient provision of civilization. Irregular eating habits due to increased stress and mental and endocrine disruptions further deepen energy imbalances. This imbalance between the energies is combined with the genetic factors of the individual to generate obesity. In the past, obesity occurred mainly in high-income countries. However, as westernization of dietary life has been spreading through economic globalization and high-calorie instant foods have been offered at a low cost worldwide, obesity patients are rapidly increasing in the middle income and low income countries. Especially in middle-income and low-income countries, which have low-calorie eating habits, the rate of increase in obese patients is becoming more severe because of the combined effect of genetic factors favoring energy accumulation.

 In 2014, consulting firm McKinsey reported that the cost of obesity is around $ 2 trillion worldwide (about $ 2230 trillion). It accounts for 2.8% of the world's gross domestic product (GDP). The cost of obesity is about the same as the cost of coping with war and terrorism, or the cost of smoking (2.1 trillion won each), alcohol ($ 1.4 trillion), illiteracy ($ 1.3 trillion) Which is much higher than the cost of responding. The Associated Press and US media quoted McKinsey as saying that obesity is not just a health issue, but a major economic challenge, and that we should take action to prevent obesity. In Korea, according to the National Health Insurance Corporation, the rate of high-altitude obesity rose from 0.2 percent in 2002 to 0.5 percent in 2013, showing an increase of 2.9 times over 11 years. The highest rate of increase in obesity rates was observed in the 20s and 30s, and women increased 6.3 times in 2013 compared to 2002, and men increased 4.8 times. Such obesity not only serves as a risk factor for overweight, but also provides an important cause of chronic adult diseases such as diabetes, hypertension, arteriosclerosis, heart disease, and joint diseases.

Obesity can be treated basically in combination with a diet that provides a low-calorie diet and exercise that increases exercise. However, since appetite is controlled by a combination of endocrine and psychological factors, it is very difficult for patients to actively control and It is also difficult to steadily exercise exercise of strength. In addition, the psychological pressure of appetite control may induce the yo-yo phenomenon, resulting in a sudden increase in body weight. Therefore, in the case of obesity caused by excessive nutrition, rather than forcing the diet and exercise to high intensity, the combination of low intensity dietary therapy and exercise therapy combined with drug treatment to suppress the fat storage of excess calories Not only can the treatment effect be expected, but also the yo-yo phenomenon can be prevented.

Drugs for the control of appetite developed in the meantime include Phentermine and Phendimetrazine which block norepinephrine secretion, Fluoxetin which blocks serotonin reuptake, and Serotonin and Norr Sibutramine, which blocks epinephrine reuptake, is a psychotropic drug that works mostly on the central nervous system. It has been recently withdrawn by the FDA because of the serious side effects such as headache, nausea and mental disorder, and depression and suicide. Orlistat, which blocks the absorption of fat in the stomach, has been reported to be less tolerant of weight loss and tolerance to long-term use, so it is urgent to develop new obesity inhibitors.

Most of the body fat is stored in fat cells. As the obesity progresses, the number of adipocytes increases numerically and accompanies a morphological change that increases in size as excessively produced adipocytes store a large amount of triglycerides. Fat cells are formed by differentiation of adipose stem cells existing in adipose tissue into adipose cells through lipid precursor cells and division and differentiation of adipose stem cells determine the number of adipocytes and the differentiation system.

According to the Department of Preventive Medicine of the Yonsei University Medical School, the adolescent obesity is very dangerous because it progresses to obesity in adult, about 14% of 6 month infant obesity, about 41% of 7 year old child obesity and about 70% of 10 ~ In fact, the prevalence of obesity in children and adolescents increased from 5.8% in 1997 to 9.6% in 2012 as a result of the National Health and Nutrition Examination Survey in 2013, and the proportion of obese, elementary, middle and high school students surveyed by the Ministry of Education also increased from 11.6% in 2006 to 15% .

The increase in the rate of obesity in children and adolescents greatly affects adult obesity morbidity because the proliferation of adipose stem cells is formed in pediatrics and adolescence and when adulthood is increased in adipose stem cell differentiation, So it affects mostly adult diseases and genetic diseases that cause fat accumulation.

In adipose tissue, not only white adipose tissue (WAT), which functions to store surplus energy in the form of triglycerides, but also the ability to maintain body temperature by generating heat in hypothermia There is brown adipose tissue (BAT) to perform.

Morphologically, white adipose cells have one large fat droplet while brown adipose cells have several small fat droplets. Brown adipocytes have a relatively large number of mitochondria compared to white adipocytes, and are brown due to the cytochrome pigment present in the mitochondria. UCP1 (uncoupling protein 1) protein is present in the mitochondrial inner membrane of brown fat cells, and UCP1 is uncoupled from the H + electrochemical concentration gradient formed during the oxidative phosphorylation of mitochondria and the ATP synthesis by ATP polymerase It releases chemical energy in the form of heat.

Small-sized mammals such as rodents are abundant in brown adipose tissue, but relatively large sized mammals such as humans have been known to lose most of the brown adipose tissue after infancy. However, recently, it has been found that a considerable amount of adipocytes showing similar characteristics to brown adipocytes are found in the supraclavicular area and neck region of adults. In particular, it was found that the UCP1 protein expression was greatly increased in the white adipose tissue when the low temperature stimulus was given, and the adipose cells showing multilocular morphological features were present in a mixed state. These adipocytes are distinct cell types not derived from the myf-5 lineage, unlike the brown adipose tissue derived from the myf-5 lineage, and named beige adipose tissue to distinguish them from typical brown adipose cells .

Beige adipocytes do not express UCP1 like white adipocytes in the absence of cold stimulation. However, when cold stimulation or cyclic AMP stimulation is given, UCP1 expression and cell respiration rate are increased to brown adipocyte level. Beige adipocytes exhibit a different gene expression pattern than white adipocytes and typical brown adipocytes and increase UCP1 expression and cellular respiration in response to irisin, a polypeptide hormone derived from muscle. Most known brown adipose cells present in adults are mostly beige adipocytes (Spiegelman BM (2013 June) Regulation of Adipogenesis: Towaed New Therapeutics for Metabolic Disease. Diabetes 62, 1774-1782; Wu et al. Jul. 20) Beige adipocytes are a distinct type of thermogenic fat cell in mouse and human. Cell 150, 366-376).

The expression of PRDM16, a transcription factor that regulates the development of brown adipocytes, was selectively expressed in adipose tissue. In the transgenic mice expressing adipose fatty acid binding protein (aP2) promoter, (17). In this study, we investigated the effects of high-density lipoprotein (HDL) -mediated adipogenesis in obesity-induced diabetic rats. 1782).

When adipogenesis is induced in mouse embryo fibroblast (MEF), white adipocytes are formed. In the same MEF, the expression of retinoblastoma protein (pRb) is inhibited or decreased Induction of adipocyte differentiation produces brown adipocytes. In addition, in the transgenic mice in which the expression of p107 protein, which is a type of Rb protein, was eliminated, the amount of white adipose tissue decreased while the amount of brown adipose tissue increased. In p107 - / - transgenic mice, pRb is present in hypophosphorylated form. These results suggest that white adipocytes and beige adipocytes differentiate from common adipose stem cell populations in adipose tissue, whereas hyperoxidized pRb induces differentiation into white adipocytes, whereas hypophosphorylated pRb induces differentiation into brown adipocytes Thereby raising the possibility of inducing differentiation. It was reported that activation of Wnt signaling at the early stage of differentiation of lipoprotein cells inhibits differentiation into brown adipocytes, but promotes differentiation into white adipocytes. Therefore, it is highly possible that interstitial differentiation into white adipocytes is inhibited from a common adipose stem cell population in adipose tissue, while intervention of drugs promoting differentiation into beige adipocytes is possible.

On the other hand, Hipk2 (homeodomain-interacting protein kinase 2) is a kinase that regulates transcription factor activity, but it has recently been reported to regulate the development of white fat, although it is involved in embryogenesis, neuron survival and apoptosis, cell proliferation and tumorigenesis . Induction of obesity by HIPK2 - / - transgenic mice with HIPK2 gene deletion resulted in a decrease in the degree of obesity and an increase in the expression of brown fat marker than in normal mice (Sjolund J et al. (2014 May 20) Identification of Hipk2 as an essential regulator of whitefat development. 111 (20): 7373-8). HIPK2 transgenic mice can be used to study the effect of fat accumulation inhibitor on adipocyte development, brown fat differentiation and fat coloration.

Accordingly, the present inventors have made intensive efforts to find a substance that inhibits the proliferation of adipose stem cells and promote fatty brownening, and as a result, it has been found that mangifera inhibits the proliferation of adipose stem cells and inhibits the differentiation of adipose stem cells into white adipocytes The present invention has been accomplished by confirming the therapeutic effect on obesity which is a cause of fat accumulation by promoting differentiation into beige adipocytes.

The main object of the present invention is to provide a method for the treatment and prophylaxis of osteogenic stem cells comprising at least one selected from the group consisting of mangiferin, neomangiferin, norathriol, and pharmaceutically acceptable salts thereof, or And to provide a composition for inhibiting proliferation or differentiation of lipid precursor cells.

Another object of the present invention is to provide a method of treating obesity, comprising administering to a mammal in need thereof an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt thereof. And to provide a composition for promoting differentiation into adipocytes or beige adipocytes.

Another object of the present invention is to provide a method for the treatment of adipocytes in a warm white color of adipose tissue comprising at least one selected from the group consisting of mangiferin, neomangiferin, norathriol, and pharmaceutically acceptable salts thereof. And a composition for inhibiting differentiation into adipocytes.

Another object of the present invention is to prevent or treat hypothermia comprising any one or more selected from the group consisting of mangiferin, neomangiferin, norathriol, and pharmaceutically acceptable salts thereof. And to provide a pharmaceutical composition for administration.

Another object of the present invention is to prevent or ameliorate hypothermia comprising any one or more selected from the group consisting of mangiferin, neomangiferin, norathriol, and a pharmaceutically acceptable salt thereof. And a food composition.

Another object of the present invention is to provide a method of controlling body temperature or body composition comprising at least one selected from the group consisting of mangiferin, neomangiferin, norathriol, and pharmaceutically acceptable salts thereof. And to provide a pharmaceutical composition for controlling energy metabolism.

Another object of the present invention is to provide a method of controlling body temperature or body composition comprising at least one member selected from the group consisting of mangiferin, neomangiferin, norathriol, And to provide a food composition for controlling energy metabolism.

Another object of the present invention is to provide a method for treating lipid metabolism-related diseases comprising at least one selected from the group consisting of mangiferin, neomangiferin, norathriol, and pharmaceutically acceptable salts thereof Or a pharmaceutically acceptable salt thereof. Such lipid metabolism related diseases may include obesity. The obesity may include childhood obesity, adolescent obesity, or adult obesity.

Another object of the present invention is to provide an anti-obesity, an appetite suppressing method comprising at least one selected from the group consisting of mangiferin, neomangiferin, norathriol, and a pharmaceutically acceptable salt thereof Inhibiting, or reducing body fat.

The composition comprising mangepiperine, neomangiectin or norathiirol according to the present invention has the effect of inhibiting the proliferation and promoting the differentiation into adipocytes of adipose or progenitor cells, It can be effectively used as a composition for cell culture for mass culture, and it is effective to inhibit the proliferation of fat stem cells and differentiate into brown fat by administration to fat-related diseases.

Accordingly, the composition comprising the mandelpepin according to the present invention can be used for prevention, improvement, and treatment of various hypothermia, lipoprotein and lipid metabolism-related diseases.

1 and 2 show the effect of mangifera on the proliferation of adipose derived mesenchymal stem cells. In Fig. 1, mangiferin was treated with adipose derived mesenchymal stem cells for 6 days, and cell proliferation was measured by MTT assay FIG. 2 shows the results of treatment of 3T3-L1 cells and HeLa cells with mangiferin for 6 days, and then cell proliferation was measured by MTT assay. Controls not treated with mangifera were labeled as control.
FIG. 3 and FIG. 4 show the effect of mangifera on adipocyte differentiation of adipose derived mesenchymal stem cells. FIG. 3 shows that adipocyte differentiation is induced by culturing in differentiation induction medium for 14 days and then stained with Oil Red O And FIG. 4 shows the measurement of absorbance at 490 nm by dissolving stained Oil Red O with isopropanol. In FIG. 3, Mangiferin (-) is a control group not treated with mangiferin, and Mangiferin (+) is treated with 10 μM of mangiferin. The X-axis represents the number of cells implanted in a 22 mm incubator. In FIG. 4, the X-axis represents the number of cells implanted in a 22 mm incubator. Controls not treated with mangifera were labeled as control.
FIGS. 5 and 6 show the effect of mangipelin on Wnt signaling in adipose derived mesenchymal stem cells. FIG. 5 shows the effect of two separate adipose derived mesenchymal stem cells and 3T3-L1 cells and HeLa cells Mannipherin was treated at a concentration of 50 μM or 100 μM for 3 days or 6 days and the amount of GSK-3β, p (Ser9) -GSK-3β, β-catenin and non-phospho-β-catenin protein was immunoblotted 6 shows the percentage of the control group in which mangiferin was not treated by measuring the p (Ser9) -GSK-3? And non-phospho-beta-catenin protein bands in Fig. 5 as a densitometer .
Figures 7 and 8 show the change in weight of an animal when the high-fat diet started in adolescence and when mangeferrin was administered. The 8-week-old adolescent animals were orally administrated for 18 weeks from the 10th week to the 28th week after the high-fat diet was started. As a result, the body weight gain was slowed compared with the control group and the difference was not changed. It remained constant. This suggests that the number of adipose stem cells proliferated in adolescence continues to be maintained until adulthood, and the weight is constantly different (Fig. 7). Feed intake was relatively constant in high fat diets compared to low fat diets (Fig. 8).
FIG. 9 and FIG. 10 show the effect of the high-fat diet on the HIPK2 gene-deficient transgenic mice that control brown lipid differentiation and the effect of the mangipiperin using the heterozygous mouse. In the 8 week-old adolescent dysplastic rat, (Fig. 9). However, the feeding amount was almost similar (Fig. 10).
FIGS. 11 and 12 show results of high-fat diet from 18 weeks to 34 weeks in order to investigate the mangiferin effect in adult rats. As a result, HIPK2 heterozygote rats weighed less at 22 weeks than normal rats and 35 weeks And mangifera, showed that only high-fat diet showed weight loss in heterozygous rats. This shows the possibility of promotion of local browning by the mangiferin in the adult. There was also some weight loss in low-fat diets.

Best Mode for Carrying Out the Invention

Based on the idea that suppression of differentiation from adipose stem cell population in adipose tissue to white adipocyte differentiation and promotion of differentiation into beige adipocytes can release surplus energy resulting from excess nutrition into heat form, The present invention has been completed upon confirming that it is effective to inhibit the differentiation of adipose stem cells into white adipocytes and to promote differentiation into beige adipocytes and to use them for the prevention or treatment of obesity.

In one aspect to accomplish the above object, the present invention provides a pharmaceutical composition comprising a compound selected from the group consisting of mangiferin, neomangiferin, norathriol, and a pharmaceutically acceptable salt thereof. The present invention provides a composition for inhibiting the proliferation or differentiation of adipose stem cells or adipose precursor cells comprising at least one adipocyte.

The above-mentioned mangiferin, neomannipiperine and norathiaryol may be represented by the following general formula (1).

The above-mentioned mangiferin, neomangiectin and norathiirol may be synthesized by a common chemical synthesis method, purchased commercially, or obtained from natural products cultivated or collected in the natural world, but are not limited thereto. For example, it may be one obtained by the method disclosed in Korean Patent No. 1097231.

The term " pharmaceutically acceptable salt " as used herein means a formulation that does not impair the biological activity and properties of the administered manipherin. The pharmaceutically acceptable salts include those acids which form a non-toxic acid addition salt containing a pharmaceutically acceptable anion such as inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, hydroiodic acid and the like, Organic carboxylic acids such as formic acid, citric acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid and salicinic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p- Sulfonic acid such as sulfonic acid, sulfonic acid, sulfonic acid, and the like. For example, pharmaceutically acceptable carboxylic acid salts include metal salts or alkaline earth metal salts formed with lithium, sodium, potassium, calcium, magnesium and the like, amino acid salts such as lysine, arginine and guanidine, dicyclohexylamine, N Organic salts such as methyl-D-glucamine, tris (hydroxymethyl) methylamine, diethanolamine, choline and triethylamine, and the like.

In the present invention, "adipose derived stem cell (ASC)" is an adult stem cell having a large amount distributed in adipose tissue, and may be obtained by isolating adipose stem cells derived from adipose tissue, but is not limited thereto .

In the present invention, "lipid precursor cells" may be used interchangeably with "adipose precursor cells, pre-adipocytes and adipose progenitor cells.

In the present invention, "differentiation" is a process in which lipid stem cells or lipid precursor cells, which are in an early stage, have characteristics as respective tissues. By the composition according to the present invention, lipid stem cells or lipid precursor cells are classified into brown fat or beige fat And can inhibit the differentiation into white fat.

In the present invention, "proliferation" is a process different from that of differentiation. When the number of adipose stem cells is increased through cell proliferation in childhood and adolescence, the adipose stem cells differentiate into adipocytes after adulthood, More than one percent will have obesity and adult disease. Therefore, inhibiting the proliferation of adipose stem cells in childhood and adolescence can prevent adult diseases including obesity.

In one embodiment of the present invention, the reduction effect of human lipid-derived mesenchymal stem cells (hAD-MSC) by mangifera is not caused by general cytotoxicity, but by the selective expression of adipocyte-derived mesenchymal stem cells (Fig. 2).

Mangiferin inhibited proliferation of adipose derived mesenchymal stem cells and promoted adipocyte differentiation similar to contact inhibition (FIGS. 3 and 4).

For example, the above-mentioned mangiferin or pharmaceutically acceptable salt thereof may be contained in an amount of 0.01 to 100% by weight, more preferably 1 to 80% by weight, based on the total weight of the pharmaceutical composition.

In another aspect, the present invention provides a method of treating an adipose tissue comprising at least one selected from the group consisting of mangiferin, neomangiferin and norathriol, or a pharmaceutically acceptable salt thereof. A composition for promoting differentiation into brown adipocytes or beige adipocytes.

In another aspect, the present invention relates to a method of treating an adipose tissue comprising at least one selected from the group consisting of mangiferin, neomangiferin, norathriol, and pharmaceutically acceptable salts thereof. A composition for inhibiting differentiation into a white adipocyte.

The above-mentioned mangiferin, or a pharmaceutically acceptable salt thereof, is as described above.

In the present invention, "brown adipose tissue (BAT)" functions to maintain body temperature by generating heat in a hypothermia state and consumes energy as heat, so that the more brown fat, . Brown adipocytes have a relatively large number of mitochondria compared to white adipocytes and are brown due to the cytochrome pigment present in the mitochondria. UCP1 (uncoupling protein 1) protein is present in the mitochondrial inner membrane of brown fat cells, and UCP1 is uncoupled from the H + electrochemical concentration gradient formed during the oxidative phosphorylation of mitochondria and the ATP synthesis by ATP polymerase It releases chemical energy in the form of heat.

The term " white adipose tissue (WAT) "refers to a fat that functions to store surplus energy in the form of triglycerides. Therefore, it is known that the inhibition of differentiation into white adipocytes is more effective for treating obesity.

In the present invention, "beige adipose tissue" is a distinct cell group not derived from the myf-5 line, unlike the brown adipose tissue derived from the myf-5 line, , But UCP1 expression and cell respiration rate are increased to the level of brown adipocytes when low temperature stimulation or cyclic AMP stimulation is given.

In the adolescent HIPK2 heterozygote rats, the high fat diet compared to the normal rats resulted in a lower body weight in the heterozygotes than in the normal rats (Fig. 9 and Fig. 10) It was confirmed that browning was accelerated (Figs. 11 and 12).

For example, the above-mentioned mangiferin or pharmaceutically acceptable salt thereof may be contained in an amount of 0.01 to 100% by weight, more preferably 1 to 80% by weight, based on the total weight of the pharmaceutical composition.

In another aspect, the present invention provides a method of preventing or reducing hypothermia comprising any one or more selected from the group consisting of mangiferin, neomangiferin, norathriol, and pharmaceutically acceptable salts thereof. A pharmaceutical composition for therapeutic use is provided.

In another aspect, the present invention provides a method of regulating body temperature, comprising administering to a mammal, including at least one selected from the group consisting of mangiferin, neomangiferin, norathriol, and pharmaceutically acceptable salts thereof, A pharmaceutical composition for regulating body energy metabolism is provided.

In another embodiment, the present invention relates to a lipid metabolism-related lipid metabolite comprising at least one member selected from the group consisting of mangiferin, neomangiferin, norathriol, and pharmaceutically acceptable salts thereof. A pharmaceutical composition for preventing or treating diseases is provided.

Such lipid metabolism related diseases may include obesity. The obesity may include childhood obesity, adolescent obesity, or adult obesity.

In addition, the lipid metabolism-related disease may be any one selected from inherited diseases including Gaucher's disease, Lehman's disease, Fabry disease, Schindler's disease, Raven's disease, sulfatase deficiency disease, Faber's disease, It is not.

In addition, the lipid metabolism-related disease may be a disease caused by hyperpolarization, hyperproliferative hyperproliferation or apoptosis of adipocytes.

The above-mentioned mangiferin, or a pharmaceutically acceptable salt thereof, is as described above.

In the cells treated with mangifera, GSK-3β activity was increased as the inactivated p (Ser9) -GSK-3β was decreased, and thus the non-phospho-β-catenin (non-phospho-beta-catenin) protein was decreased. Thus, mangiferin enhances the activity of the Wnt /? -Catenin signaling pathway by inhibiting GSK-3 ?, suggesting that mannipurein has a therapeutic effect on obesity-related lipid metabolism-related diseases.

As used herein, the term "prevention" may refer to any action that inhibits or delays the onset of a disease by administering the mangeferin according to the present invention to the individual.

As used herein, the term "treatment" may refer to any act which, by administering the composition of the present invention to a suspected disease susceptible individual, may improve or improve symptoms of the disease.

The pharmaceutical composition of the present invention can be used as a single preparation and can be used as a combined preparation containing a drug known to have an effect of treating hypothermia, temperature control, body energy metabolism regulation, or lipid metabolism related disease, May be prepared in unit dose form or formulated into multi-dose containers by formulating them with pharmaceutically acceptable carriers or excipients.

As used herein, the term "pharmaceutically acceptable carrier" may mean a carrier or diluent that does not disturb the biological activity and properties of the compound being injected, without irritating the organism. The type of the carrier that can be used in the present invention is not particularly limited, and any carrier conventionally used in the art and pharmaceutically acceptable may be used. Non-limiting examples of the carrier include saline, sterilized water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and the like. These may be used alone or in combination of two or more. The carrier may comprise a non-naturally occuring carrier.

In addition, if necessary, other conventional additives such as an antioxidant, a buffer and / or a bacteriostatic agent can be added and used. A diluent, a dispersant, a surfactant, a binder, a lubricant, Pills, capsules, granules or tablets, and the like.

In addition, the pharmaceutical composition of the present invention may contain a pharmaceutically effective amount of mangifera. The term "pharmaceutically effective amount" as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and is generally in the range 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 a day to several times per day. For purposes of the present invention, however, the specific therapeutically effective amount for a particular patient will depend upon the nature and extent of the reaction to be achieved, the particular composition, including whether or not other agents are used, the age, weight, Sex and diet of the patient, the time of administration, the route of administration and the rate of administration of the composition, the duration of the treatment, the drugs used or concurrently used with the specific composition, and similar factors well known in the medical arts.

The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with another therapeutic agent, and may be administered sequentially or simultaneously with conventional therapeutic agents. And can be administered singly or multiply. It is important to take into account all of the above factors and to administer an amount that can achieve the maximum effect in a minimal amount without causing side effects, and can be readily determined by those skilled in the art.

The term "administering" as used herein refers to the introduction of a pharmaceutical composition of the present invention to a patient by any suitable method, and the route of administration of the composition of the present invention may be oral or parenteral May be administered via various routes.

The mode of administration of the pharmaceutical composition according to the present invention is not particularly limited and may be conventionally used in the art. As a non-limiting example of such a mode of administration, the compositions may be administered orally or parenterally. The pharmaceutical composition according to the present invention can be manufactured into various formulations according to the intended administration mode.

The frequency of administration of the composition of the present invention is not particularly limited, but it may be administered once a day or divided into several doses.

In another aspect, the present invention provides a method of preventing or treating hypothermia or a lipid metabolism-related disease comprising administering the above-mentioned mangiferae to a subject.

As used herein, the term "individual" may refer to any animal, including humans, who have or are at risk of developing hypothermia or lipid metabolism related diseases. The animal may be, but is not limited to, a mammal such as a cow, a horse, a sheep, a pig, a goat, a camel, a nutrient, a dog, a cat,

The preventive or therapeutic method of the present invention may specifically include administering the composition in a pharmaceutically effective amount to a subject suffering from or at risk of developing hypothermia or a lipid metabolism related disease.

The term "administering" as used herein refers to the introduction of a pharmaceutical composition of the present invention to a patient by any suitable method, and the route of administration of the composition of the present invention may be oral or parenteral May be administered via various routes.

In another aspect, the present invention provides a method of preventing or reducing hypothermia comprising any one or more selected from the group consisting of mangiferin, neomangiferin and norathriol, or a pharmaceutically acceptable salt thereof, There is provided a food composition for improvement.

Hypothermia can mean a state in which the central body temperature (deep body temperature) falls below 35 ° C. Since hypothermia is caused when the body's heat production is reduced, when heat loss is increased, or when the two occur in combination, the composition of the present invention promotes brown localization to generate heat in the hypothermia state to maintain body temperature .

In another aspect, the present invention provides a method of regulating body temperature comprising the administration of at least one selected from the group consisting of mangiferin, neomangiferin, and norathiirol, or a pharmaceutically acceptable salt thereof, A food composition for regulating body energy metabolism is provided.

The body temperature control may mean a regulating action in which an individual maintains the body temperature in the optimal range to match the activity. The composition of the present invention promotes brown localization to generate heat to maintain body temperature, thereby effectively controlling body temperature.

In another aspect, the present invention provides a composition comprising an anti-obesity agent comprising one or more selected from the group consisting of mangiferin, neomangiferin and norathiirol, or a pharmaceutically acceptable salt thereof, A food additive, an appetite suppressing agent, or a body fat reducing agent.

The food composition may include a health functional food.

Since the above-mentioned mangifera, neomangiectin and norathiirol have been used for a long time as a natural substance and proved to be stable, they are manufactured in the form of food which is common but can prevent or improve obesity, inhibit appetite, or reduce body fat It can be ingested.

Functional food is the same term as food for special health use (FoSHU). It refers to foods that have been processed so that the biomechanical functions are efficiently displayed in addition to the nutritional supply. , The food may be prepared in various forms such as tablets, capsules, powders, granules, liquids, rings and the like in order to obtain the effect of preventing or improving obesity, inhibiting appetite, or reducing body fat.

The food composition of the present invention may further comprise a pharmaceutically acceptable carrier.

There is no particular limitation on the kind of the food to which the composition containing the mangifera, neomangiopherin, and / or norathiirol of the present invention can be added. For example, various beverages, gums, tea, vitamins, . The food composition may be supplemented with other ingredients that do not interfere with the prevention or amelioration of obesity, an appetite suppression effect, or a body fat reduction effect, and the kind thereof is not particularly limited. For example, various herbal medicine extracts, food-acceptable food-aid additives or natural carbohydrates, such as ordinary foods, may be added as an additional ingredient.

The food-aid additive is added to produce a health functional food of each formulation and can be appropriately selected and used by those skilled in the art. For example, various kinds of nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and fillers, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, , A stabilizer, a preservative, a glycerin, an alcohol, a carbonating agent used in a carbonated drink, and the like, but the kind is not limited by the above examples.

At this time, the content of the extract contained in the food is not particularly limited, but may be 0.01 to 100% by weight, more preferably 1 to 80% by weight based on the total weight of the food composition.

When the food is a beverage, it may be contained in a proportion of 1 to 30 g, preferably 3 to 20 g based on 100 ml. In addition, the composition may contain additional ingredients which are commonly used in food compositions and which can improve odor, taste, vision and the like. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, panthotenic acid and the like. In addition, it may include minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn) and copper (Cu) It may also include amino acids such as lysine, tryptophan, cysteine, valine, and the like. In addition, it is also possible to use antiseptics (such as potassium sorbate, sodium benzoate, salicylic acid, and sodium dehydroacetate), disinfectants (such as bleaching powder and highly bleached white powder, sodium hypochlorite), antioxidants (butylhydroxyanilide (BHA), butylhydroxytoluene BHT), etc.), coloring agents (such as tar pigments), coloring agents (such as sodium nitrite and sodium acetic acid), bleaching agents (sodium sulfite), seasoning (such as MSG sodium glutamate), sweeteners (such as hypoglycemia, , Food additives such as flavorings (vanillin, lactones, etc.), swelling agents (alum, potassium hydrogen D-tartrate), emulsifiers, emulsifiers, thickeners, encapsulating agents, gum bases, foam inhibitors, ) Can be added. The additives are selected according to the type of food and used in an appropriate amount.

The health functional food of the present invention can be manufactured by a method commonly used in the art and can be prepared by adding raw materials and ingredients which are conventionally added in the art. Also, unlike general medicine, there is an advantage that there is no side effect that can occur when a medicine is used for a long time by using food as a raw material, and it is excellent in portability.

The pharmaceutically acceptable salt may be used in the same sense as the pharmaceutically acceptable salt.

HIPK2 +/- heterozygous transgenic mice were not significantly different from normal rats in obesity induced by high fat diet, but the degree of obesity was decreased when we administered mangifera. This suggests that mangeferrin may promote brown fat differentiation in the adipose tissue formation of adult by high fat diet. In addition, the increase in obesity was significantly different in the high-fat diabetic rats fed 8-week-old high-fat diet rats, This suggests that mangiferin inhibits proliferation of adipose stem cells in adipose tissue by high - fat diets, thus greatly suppressing adolescent obesity.

In another aspect, the present invention provides a composition comprising an anti-obesity agent comprising one or more selected from the group consisting of mangiferin, neomangiferin and norathiirol, or a pharmaceutically acceptable salt thereof, A food composition for reducing appetite, or for reducing body fat.

The feed composition may include a feed additive. The feed additive of the present invention corresponds to an auxiliary feed in the feed control method.

The term "feed" as used herein in the context of the present invention may mean any natural or artificial diet, single meal, or the like ingredients for feeding, ingesting, digesting or suitable for the animal.

The kind of the feed is not particularly limited, and feeds conventionally used in the art can be used. Non-limiting examples of such feeds include vegetable feeds such as cereals, muscle roots, food processing busines logistics, algae, fibers, pharmaceutical buses, oils, fats, pastes or grain by-products; Animal feeds such as proteins, inorganic substances, fats, oils, fats, oils, monocellular proteins, animal plankton or foods. These may be used alone or in combination of two or more.

In another aspect, the present invention provides quasi-compositions for anti-obesity, appetite suppression, or body fat reduction.

The term "quasi-drug" in the present invention means a fiber, a rubber product or the like used for treating, alleviating, treating or preventing a disease of a human or an animal, Or products similar to those that are not machinery, preparations used for sterilization, insecticides and similar uses for the prevention of infectious diseases, for the purpose of diagnosing, treating, alleviating, treating, or preventing diseases of human beings or animals Means an article, except machinery, machinery or equipment, of the commodity used, and not used for the purpose of giving pharmacological effects to the structure and function of humans or animals; The quasi-drug may also include external preparations for skin and personal hygiene products. But is not limited to, a disinfectant cleaner, a shower foam, a guarine, a wet tissue, a detergent soap, a hand wash, or an ointment.

When the composition according to the present invention is used as a quasi-drug additive, the composition may be added as it is or may be used together with other quasi-drugs or quasi-drugs, and may be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be appropriately determined depending on the purpose of use.

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited by the following examples.

Example 1. Preparation of materials

Dulbecco's modified eagle media-F12 medium, Ca + 2 / Mg + 2-free HBSS (Hank's balanced salt solution), trypsin-ethylenediamine tetraacetic acid, secondary antibodies and L- glutamine, (Carlsbad, CA, USA). Primary antibodies were purchased from Abcam (Cambridge, UK), Chemicon (Billerica, MA, USA) and Serotec (Kiddlinton, UK). Secondary antibodies were purchased from Jackson (West Grove, PA, USA). Piperazineethanesulfonic acid, phenol red, immunoprecipitation assay buffer, phosphate buffered saline (PBS), poly-L-ornithine, insulin, fibronectin, 4- (2-hydroxyethyl) , A phosphatase inhibitor mixture, a protease inhibitor mixture, dimethyl sulfoxide, paraformaldehyde (PFA) and Tween 80 were purchased from Sigma (St. Louis, MO, USA).

Mangiferin (purity 95% or more) is reported by Kangsik JUNG et al. Pharm. Bull. 32 (2) 242-46, 2009). ≪ tb > < TABLE > All other materials were obtained from standard commercial sources.

Example 2 Isolation and Culture of Human Adipose-derived Mesenchymal Stem Cells (hAD-MSC)

The human adipose-derived mesenchymal stem cells were treated with collagenase I for 30 minutes at 37 ° C, filtered through a cell strainer, and centrifuged at 1,500 rpm for 10 minutes to obtain 10% heat And cultured in DMEM medium supplemented with inactivated FBS at 5% CO 2 and 37. The surviving cells were treated with 2.5% trypsin-EDTA to remove the cells and cultured in Mesen PRO medium supplemented with 2 mM glutamax (5% CO ₂ , 37) (primary culture).

Example 3: Effect of mangifera on the proliferation of adipose derived mesenchymal stem cells

In Example 2, the primary cultured human adipose-derived mesenchymal stem cells were planted in 2x10 ⁴ cells in a 35 mm incubator. DMEM medium supplemented with 10% FBS, 2 mM glutamax, 5 μg / μl gentamicin, and 10 ng / ml EGF was used. To confirm the effect of mangifera on the proliferation of human adipose derived mesenchymal stem cells, mangifera was added to the culture medium at concentrations of 0.01, 0.1, 1, 10, 50, and 100 μM. After 6 days of incubation, the culture was replaced with 1 ml of fresh culture medium and 100 μl MTT solution was added. After incubation for 4 hours at 37 ° C in 5% CO2, the culture medium was removed. The resulting formazan crystals were dissolved in 2 ml of DMSO (dimethyl sulfoxide), and the absorbance at 550 nm was measured.

When adipocyte mesenchymal stem cells were treated with mangifera, the cell proliferation was reduced in a concentration-dependent manner (FIG. 1). The cell proliferative effect was observed at a concentration of 1 μM of mangiferin and decreased to 63% of that of the control group. At 50 μM and 100 μM concentrations, the cell proliferative effect was almost saturated and decreased to 50% of that of the control group.

To investigate the cell selectivity of the inhibitory effect of mangifera on cell proliferation, the effect of mangifera on cell proliferation of 3T3-L1 adipocytes and HeLa cells as uterine cancer cells was investigated (Fig. 2). The cell proliferation of 3T3-L1 and HeLa cells was not different from that of the control group even when treated with 100 μM of mangifera as the highest concentration.

Therefore, it was confirmed that the effect of hansenferin-induced reduction of hAD-MSC cell proliferation is not caused by general cytotoxicity, but is an activity selectively appearing in adipose-derived mesenchymal stem cell proliferation.

Example 4. Effect of Manipiperin on adipogenesis of adipose derived mesenchymal stem cells

Cells were cultured for 1 day in the basal medium and then replaced with differentiation induction medium supplemented with 1 μM dexamethasone, 0.5 mM isobutylmethylxanthine, 0.2 mM indomethacin, and 10 μM human insulin in the basic medium. Lt; / RTI > Differentiation induction medium was replaced every 3 days.

Then, 50 μM or 100 μM mannipherin was added to the culture medium to analyze the effect of mangifera on adipocyte differentiation of adipose derived mesenchymal stem cells. Cells were seeded at a density of 0.4x10 ⁵ , 0.8x10 ⁵ , and 1.6x10 ⁵ in a 22 mm incubator. After 14 days of culture, the cells were stained with Oil Red O and observed. The stained fat droplets were dissolved in isopropanol and absorbance was measured at 490 nm.

In all cases cultured at three densities, the experimental group treated with mangiferin showed 15% to 40% increase in Oil Red O staining compared to the control group, thereby confirming that adipocyte differentiation was effectively promoted (FIGS. 3 and 4). In order for mesenchymal stem cells to differentiate into adipocytes, differentiation stimuli must be provided after cell cycle progression is stopped by contact inhibition. As shown in Example 3 above, it was confirmed that mangiferae inhibited proliferation of adipose derived mesenchymal stem cells and promoted adipocyte differentiation similar to contact inhibition.

Example 5. Effect of Manipiperin on Wnt signaling in adipose derived mesenchymal stem cells

Embodiment planted primary culture of human adipose derived Mesenchymal Stem Cells In Example 2 to 6x10 ⁴ cells in 60mm culture medium. Mangiferin was added to the culture at concentrations of 0, 50 and 100 μM. After culturing for 3 or 6 days, the cell extract was obtained by dissolving in RIPA buffer solution. Proteins were quantitatively analyzed by Bradford protein analysis, and the same amount of proteins were separated by SDS-polyacrylamide gel electrophoresis. Proteins were transferred to nitrocellulose membrane and immunoblotting experiments were performed. Pan-GSK-3β, p (Ser9) -GSK-3β, pan-β-catenin and non-phospho-β-catenin antibodies were used as primary antibodies. The pan-GSK-3β antibody recognizes all GSK-3β proteins and the p (Ser9) -GSK-3β antibody recognizes only the inactivated GSK-3β protein. The pan-β-catenin antibody recognizes all β-catenin proteins and the non-phospho-β-catenin antibody recognizes only the activated β-catenin protein.

GSK-3β protein phosphorylates β-catenin and ultimately removes β-catenin from cells. Cells treated with mangifera at a concentration of 50 [mu] M and 100 [mu] M decreased the amount of p (Ser9) -GSK-3 [beta] protein compared to the control group (Fig. 5 and Fig. 6). The higher the concentration of mangiferae treated, the greater the decrease in p (Ser9) -GSK-3β protein in the cells treated for 6 days than in the cells treated for 3 days. In addition, the amount of non-phospho-β-catenin protein decreased in the mangifera treated cells compared to the control. The concentration of non-phospho-β-catenin was decreased in the cells treated for 6 days compared to the cells treated for 3 days, -3 < / RTI > protein.

That is, in the cells treated with mangifera, the GSK-3β activity was increased as the inactivated p (Ser9) -GSK-3β was decreased, and thus the non-phospho-β- catenin protein was decreased.

Similar results were observed in the separately obtained human adipose derived mesenchymal stem cells. In the 3T3-L1 cells and HeLa cells without inhibition of cell proliferation by mangifera, the amount of p (Ser9) -GSK-3 and non-phospho-β-catenin protein was significantly different between the experimental group treated with mangepiperine and the control group There was no difference.

As a result, mangiferin enhances the activity of the Wnt /? -Catenin signaling pathway by inhibiting GSK-3 ?, suggesting that mannipurein has a therapeutic effect on obesity-related lipid metabolism-related diseases.

Example 6. Preparation of a mouse model of a high fat diet and administration of manzipherin

(1) Preparation of animals

Male rats (30 g) and high-fat diet were purchased from the Orient Animal Breeding Center (Branch of Charles River Laboratories, Gyeonggi-do, Korea). Rats were housed four or five per cage during at least one week of acclimation and were fed with water and food (ad libitum) and maintained at constant temperature (20-23 ± 1), humidity (60 ± 10% 12h / 12h) cycle method (light 07: 00-19: 00 o'clock) condition was maintained. Obese rats were divided into two or three cages per cage to avoid social stress. All experiments followed NIH and Kyung Hee University animal care and use guidelines.

(3) oral administration of mangepiperine

The control group was orally administered with the carrier (10% DMSO) and the mangifera group dissolved in DMSO at 20 mg / kg in the manipherin group. Carriers or mangepirin groups were orally administered to the upper part of the mouth by using an oral injection needle (oral zodiac needle, 5 cm) once daily for 1 ml, and no vomiting reaction was observed.

(3) Adolescent animals are given high-fat diet

To investigate animal weight changes and brown fat differentiation due to decreased proliferation of adipose stem cells when mugipherin was administered at the beginning of adolescence, high-fat diet was started for 8-week-old adolescents, Weighed and feed intake. From 10 to 28 weeks, manganese perine was orally administered for 18 weeks. Compared to the saline-DMSO vehicle immediately after the administration of mangifera (HFD-MGF, N = 5), the difference in body weight remained relatively constant until 28 weeks after the start of weight difference. This suggests that the number of adipose stem cells proliferated in adolescence continues to be maintained until adulthood, and the weight is constantly different (Fig. 7). Feed intake was relatively constant in the case of high fat diets compared to low fat diets (Fig. 8).

Example 7. Study of HIPK2 Transgenic Rats Regulating Brown Fertilization

(1) Preparation of animals

Female experimental rats (18 g) and high-fat diet were purchased from the Orient Animal Breeding Center (Branch of Charles River Laboratories, Gyeonggi-do, Korea). Rats were housed four or five per cage during at least one week of acclimation and were fed with water and food (ad libitum) and maintained at constant temperature (20-23 ± 1), humidity (60 ± 10% 12h / 12h) cycle method (light 07: 00-19: 00 o'clock) condition was maintained. Obese rats were divided into two or three cages per cage to avoid social stress. All experiments followed NIH and Kyung Hee University animal care and use guidelines.

(2) HIPK2 Gene Elimination Transgenic mice were treated with high fat diet

HIPK2 gene deletion transgenic mice were reported to have a decrease in white fat and brown coloration in homozygous rats and slight differences in females in heterozygotes. In order to investigate the effect of mangifera on heterozygous rats, we firstly showed that adolescent dysplastic zygote rats weighing less than those in normal rats were more likely to be fed high fat diets compared to normal rats. However, in the low fat diet, there was almost no difference in the weight of the heterozygous and normal mice (Fig. 9). However, the feeding amount was almost similar (Fig. 10).

 (3) Administration of high-fat diet to adult HIPK2 heterozygous rats

To investigate the effects of the mangiferin on adult rats, high - fat diets were fed from 18 to 34 weeks of age. The body weights of HIPK2 heterozygote rats were lower than those of normal rats at 22 weeks of age. The weight did not increase but remained almost constant. At 35 weeks of treatment with mangifera, weight loss was observed only in heterozygous rats. This seems to be the result of accelerated lipid brownening by mangiferaine in HIPK2 heterozygous rats. In low-fat diets, some weight loss was also observed in normal rats (Figs. 11 and 12).

These results suggest that the lipid - lowering effect of mangiferin is significantly reduced by adipocyte reduction in adolescents and decreased by fatty brownening in adults.

The composition comprising mangepiperine according to the present invention can be used for prevention, improvement and treatment of various hypothermia, lipoprotein and lipid metabolism-related diseases.

Claims (11)

Proliferation or proliferation of adipose stem cells or adipose precursor cells comprising any one or more selected from the group consisting of mangiferin, neomangiferin, norathriol, and pharmaceutically acceptable salts thereof, or Composition for inhibiting differentiation. Brown fat cells or beige fat cells of adipose tissue comprising any one or more selected from the group consisting of mangiferin, neomangiferin, norathriol, and pharmaceutically acceptable salts thereof. Wherein the composition for promoting differentiation into a plant is a plant. For inhibiting the differentiation of adipose tissue into white adipocytes comprising any one or more selected from the group consisting of mangiferin, neomangiferin, norathriol, and pharmaceutically acceptable salts thereof. Composition. A pharmaceutical composition for preventing or treating hypothermia comprising at least one selected from the group consisting of mangiferin, neomangiferin, norathriol, and a pharmaceutically acceptable salt thereof. A food composition for preventing or ameliorating hypothermia comprising any one or more selected from the group consisting of mangiferin, neomangiferin, norathriol, and a pharmaceutically acceptable salt thereof. A pharmaceutical composition for controlling body temperature or controlling body energy metabolism comprising at least one selected from the group consisting of mangiferin, neomangiferin, norathriol, and pharmaceutically acceptable salts thereof. A food composition for regulating body temperature or regulating body energy metabolism comprising at least one selected from the group consisting of mangiferin, neomangiferin, norathriol, and a pharmaceutically acceptable salt thereof. For the prevention or treatment of lipid metabolism-related diseases comprising any one or more selected from the group consisting of mangiferin, neomangiferin, norathriol, and pharmaceutically acceptable salts thereof. Composition. 9. The pharmaceutical composition according to claim 8, wherein the lipid metabolism-related disease comprises obesity. 10. The method of claim 9,
Wherein said obesity is childhood obesity, adolescent obesity, or adult obesity. An anti-obesity, an appetite suppressant, or a body fat reducing agent, which comprises any one or more selected from the group consisting of mangiferin, neomangiferin, norathriol, and a pharmaceutically acceptable salt thereof Food composition.

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