WO2013085338A2 - Pharmaceutical composition for preventing and treating metabolic diseases, comprising nymphaea tetragona root extract, fractions thereof, or polyphenol-based compounds isolated from the nymphaea tetragona root extract as active ingredients - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing and treating metabolic diseases, comprising nymphaea tetragona root extract, fractions thereof, or polyphenol-based compounds isolated from the nymphaea tetragona root extract. The nymphaea tetragona root extract and fractions thereof effectively inhibit the activity of glycerol-3-phosphate (GPAT), which may cause metabolic diseases, and exhibit inhibitory activity against the biosynthesis of neutral fats in cells and increased insulin-dependent glucose uptake activity in skeletal muscle cells. Thus, nymphaea tetragona root extract, fractions thereof, or polyphenol-based compounds isolated from the nymphaea tetragona root extract can be effectively used in the prevention and treatment of metabolic diseases such as obesity, type 2 diabetes, dyslipidemia, insulin resistance, hepatic steatosis, and non-alcoholic fatty liver disease.

Description

 【Specification】

 [Name of invention]

 Pharmaceutical composition for the prevention and treatment of metabolic diseases containing water lily extract, fractions thereof or polyphenol-based compounds isolated therefrom as active ingredients

Technical Field

 The present invention provides obesity, type 2 diabetes mellitus, dyslipidemia, insulin resistance, hepatic steatosis, and non-alcoholic activity containing water extract extract, fractions thereof, or polyphenol-based compounds isolated therefrom as active ingredients. The present invention relates to a pharmaceutical composition for preventing and treating metabolic diseases such as fatty liver. .

Background Art

 The triglyceride biosynthesis is a multistage reaction, the first of which is catalyzed by glycerol-3-phosphate acyl transferase (GPAT) and the glycerol 3-phosphate of glycerol 3-phosphate. The fatty acid (fatty acyl-CoA), a form of acyl-coenzyme A, is transferred to the STT 1 position to catalyze the ester reaction of producing lysophosphatidic acid OysophosphaticHc acid (LPA) and glycerol-3 It is known as the rate-1 imiting step that regulates the rate of the glycerol-3-phosphate pathway (Be 11 et al, Annu Rev Biochem 49: 459-487, 1980). The final step is the biosynthesis of triglycerides by diacylglycerol acyl transferase (DGAT).

To date, GPAT has four types of isoenzymes in mammalian tissues and is distinguished by their sensitivity to N-ethylmaleimide (NEM) and their location. In other words, GPAT3 and GPAT4 present in the endoplasmic reticulum (ER) exhibit high sensitivity to sulfhydryl reagents containing NEM and mitochondrial outer membrane. MtGPATl and mtGPAT2 present in the membrane (MOM) are resistant to NEM and have a characteristic of favoring saturated fatty acyl-CoO as a substrate (Gonzanlez-Baro et al. Biochim. Biophys. acta, 1771: 830-). 838, 2007) mtGPAT accounts for about 10% of total GPAT activity in most tissues of the human body, but up to 50% of total GPAT activity in liver tissues and high activity in adipose tissues. (Bell et al, Annu Rev Biochem 49: 459-487, 1980; Lewin et al. Arch. Biochem. Biophys. 396: 119-127, 2001), suggesting that mtGPAT is an important component of fatty liver formation, obesity and insulin resistance treatment. It can be a useful target.

 Of the four isoforms of GPAT, only mtGPAT is known to be affected by diet and exercise. Studies have shown that when high calorie diets can be used to consume excess calories, the mRNA expression of mtGPAT is increased and the activity of mtGPAT is increased as a result. In the mice group, mtGPAT activity was steadily increased compared with mice without exercise at all, resulting in a significant increase in triglyceride synthesis (KuMp et al. 2006).

 Selective overexpression of mtGPAT in rats in rats increased lipid biosynthesis in liver, significantly reduced fatty acid oxidation, and increased fatty liver, dyslipidemia, and insulin resistance in rats. In particular, insulin resistance in the liver was greatly increased (Linden et al. FASEB J, 20: 434-443, 2006; Linden et al. J. Lipid Res. 45: 1279-1288, 2004). This means that lipid metabolism in the liver is mainly involved in the development of insulin resistance. In particular, the high incidence of fatty liver in obese patients suggests that mtGPAT may be an attractive molecular target for the development of therapeutic agents for dyslipidemia and insulin resistance associated with non-alcoholic fatty liver disease. (Linden et al., FASEB J, 20: 434—443, 2006).

In a related study, induction of obesity in mtGPAT-deficient (mtGPAT——) mice If you eat a high fat, high sugar diet,

CPT-1 and β—oxidation were activated, and mtGPAT-deficient (mtGPAT—) mice had 37¾ less triglyceride in the liver and 15% less blood than the control group. The secretion of very low density lipoprotein (VLDL) was reduced by 30%. As a result, mtGPAT ᅳ /-was observed in mice to increase insulin sensitivity after weight and fat loss (Hammond et al, Mol Cell Biol 22). : 8024-8214, 2002; Hammond et al, J Biol Chem 280: 25629x25636, 2005). Therefore, GPAT inhibition may be a function of inhibiting intracellular accumulation of triglycerides, and the development of low molecular weight inhibitors that regulate the fat accumulation and energy metabolic role of GPAT has been associated with obesity, type 2 diabetes, dyslipidemia, insulin resistance, It may be a strategy to develop therapeutic agents for metabolic diseases such as hepatic steatosis and non-alcoholic fatty liver.

Conventional mtGPAT inhibitor compounds include cyclopentenyl acetic acid ^ 010 6] ^ 1 ^ 1 acetic acid derivatives (Eydysh et al. Bioorg. Med. Chem. 2010, 18: 6470-6479), 2- (nonylsulfonamido) and benzoic acid (benzoic acid) derivatives (Eydysh et al. J. Med. Chem. 2009, 52: 3317-3327) are known, but exhibited weak inhibitory activity as enzyme inhibitors with IC ₅₀ values between 25 μΜ and 350 μΜ. The enzyme source used was interpreted as a mitochondrial fraction of ¾, which is still in its infancy. Patent No. 1, in which the genetic inhibition of mtGPAT and lipid metabolism-related enzymes in collaboration with FASgen and Princeton University of the United States, is effective in the inhibition and treatment of various viral infections is known (TO 2011/019498).

 On the other hand, it is a plant belonging to ^ Nyi haea tetragona) ^ water lily family, which is distributed in Korea, Japan, China, India, East Siberia, and many sea stalks are aquatic plants, and many petioles grow on the short and short stems of flowers and bloom on the water. The flower of water lily is used to soothe the heat in one shot, to loosen the hangover, and to treat acute puberty in children. In the folk, flowers are used for drugs such as hemostasis, tonic, and insomnia.

 Geraniin, a polyphenolic compound,

1,2,3,4, 6-pentazol 0-galoi-betabeta-glucose (1,2,3,4, 6-pent a ^to 0-ga 11 oyl-bet aDg 1 ucose, PGG) is known Radiation induced cell death (apoptosis) It is known to inhibit (Kanget al. Cell Biol. Toxicol. 2011, 27: 83-94; Biol. Pharm. Bull. 2010, 33: 1122-1127). The present inventors effectively inhibited the enzyme activity of mtGPATl in the process of searching for active substances that inhibit ratGPATl from natural products, and inhibited the biosynthesis of triglycerides in cells. By confirming that glucose intake is increased in skeletal muscle cells, it is effective in preventing and treating metabolic diseases such as obesity, type 2 diabetes, dyslipidemia, insulin resistance, hepatic steatosis and nonalcoholic fatty liver. The invention has been completed.

[Detailed Description of the Invention]

 [Technical problem]

 An object of the present invention is to provide a pharmaceutical composition for the prevention and treatment of metabolic diseases containing water lily root 0V7¾oA3es tetragona) extract as an active ingredient.

 In addition, another object of the present invention is to provide a pharmaceutical composition for preventing and treating metabolic diseases, comprising a fraction obtained by fractionating the water lily root extract using an organic solvent.

 In addition, another object of the present invention is a pharmaceutical composition for preventing and treating metabolic diseases comprising a polyphenol-based compound of Formula 1, Formula 2, Formula 3 or Formula 4, or a pharmaceutically acceptable salt thereof as an active ingredient. To provide.

<Formula 1>

<Formula 3>

 <Formula 4>

아을러, 본 발명의 다른 목적은 수련뿌리 추출물， 이의 분획물 또는 이로부터 분리된 폴리페놀계 화합물을 유효성분으로 함유하는 대사성 질환 예방 및 개선용 건강식품용 조성물을 제공하는 것이다.

In addition, another object of the present invention to provide a health food composition for preventing and improving metabolic diseases containing water lily root extract, fractions thereof or polyphenol-based compounds isolated therefrom as an active ingredient.

Technical Solution

 In order to solve the above problems, the present invention provides a pharmaceutical composition for the prevention and treatment of metabolic diseases containing water lily root (vVj / ^ ea tetragona) extract as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing and treating metabolic diseases, including a fraction obtained by fractionating the water lily root extract using an organic solvent. In addition, the present invention is a polyphenol-based compound of Formula 1, Formula 2, Formula 3 or Formula 4, or a pharmaceutically acceptable salt thereof as an active ingredient Provided is a pharmaceutical composition for preventing and treating metabolic diseases.

 The present invention also provides a method for treating metabolic disease comprising administering a pharmaceutically effective amount of a water lily root extract to an individual suffering from metabolic disease.

 In addition, the present invention provides a method of preventing metabolic diseases comprising administering to a subject a pharmaceutically effective amount of a water lily root extract.

 The present invention also provides a method of treating metabolic disease comprising administering a pharmaceutically effective amount of an organic solvent fraction of water lily root extract to an individual suffering from metabolic disease.

 In addition, the present invention provides a method for preventing metabolic diseases comprising the step of throwing a pharmaceutically effective amount of an organic solvent fraction of water lily root extract to an individual.

 In addition, the present invention comprises administering a pharmaceutically effective amount of the polyphenol-based compound of Formula 1, Formula 2, Formula 3 or Formula 4, or a pharmaceutically acceptable salt thereof to an individual suffering from metabolic disease. It provides a method of treating metabolic diseases.

 In addition, the present invention provides a method for treating a metabolic disease comprising administering to a subject a pharmaceutically effective amount of the polyphenolic compound of Formula 1, Formula 2 or Formula 3 or Formula 4, or a pharmaceutically acceptable salt thereof. Provide preventive measures.

 In another aspect, the present invention provides a composition for preventing and improving metabolic diseases containing water lily extract as an active ingredient. ^

 In addition, the present invention provides a composition for preventing and improving metabolic diseases, including a fraction obtained by fractionating the water lily root extract using an organic solvent.

In addition, the present invention provides a composition for preventing and improving metabolic diseases, including the polyphenol-based compound of Formula 1, Formula 2, Formula 3 or Formula 4, or a pharmaceutically acceptable salt thereof as an active ingredient. do. The present invention also provides a composition containing a water lily root extract for use in the prevention and treatment of metabolic diseases.

 The present invention also provides a composition containing an organic solvent fraction of water lily root extract for use in the prevention and treatment of metabolic diseases.

 The present invention also provides a polyphenol-based compound of Formula 1, Formula 2, Formula 3 or Formula 4, or a pharmaceutically acceptable salt thereof, for use in the prevention and treatment of metabolic diseases.

 The present invention also provides the use of water lily root extract for use in the preparation of pharmaceutical compositions for the prevention and treatment of metabolic diseases.

 The present invention also provides the use of an organic solvent fraction of a water lily root extract for use in the preparation of a pharmaceutical composition for the prevention and treatment of metabolic diseases.

 In addition, the present invention provides a use of the polyphenol-based compound of Formula 1, Formula 2, Formula 3 or Formula 4, or a pharmaceutically acceptable salt thereof for use in the preparation of a pharmaceutical composition for preventing and treating metabolic diseases. do.

 In addition, the present invention provides the use of water lily root extract for use in the manufacture of a composition for the prevention and improvement of health foods for metabolic diseases.

 In addition, the present invention provides the use of the organic solvent fraction of the water lily root extract for use in the preparation of the composition for the health food for preventing and improving metabolic diseases. In addition, the present invention provides a use of the polyphenol-based compound of Formula 1, Formula 2, Formula 3 or Formula 4, or a pharmaceutically acceptable salt thereof for use in the preparation of a health food composition for the prevention and improvement of metabolic diseases to provide.

Advantageous Effects

Water lily root extract, extracts thereof or polyphenolic compounds isolated from the fractions according to the present invention effectively inhibit the activity of mtGPATl causing metabolic disease, inhibit the biosynthesis of intracellular triglycerides and insulin-dependent in skeletal muscle cells As it was confirmed that it showed the activity to increase glucose intake, Including the polyphenolic compounds of the present invention, water extracts or their fractions containing them are useful for the prevention and treatment of metabolic diseases such as obesity, type 2 diabetes, dyslipidemia, insulin resistance, hepatic steatosis and nonalcoholic fatty liver. Can be used.

[Brief Description of Drawings]

 1 and 2 is a diagram showing the inhibitory activity of hGPATl of the water extract methanol extract, butanol fraction and the compound isolated from the fraction.

 1 is a diagram showing the inhibitory activity of hGPATl of the water extract methanol extract and solvent fractions.

 Figure 2 is a diagram showing the hGPATl inhibitory activity of the formula (1), (2), (3) or (4).

 3 is a diagram showing the effect of inhibiting triglyceride biosynthesis in HepG2 cells (human-derived hepatocytes) of the methanol extract and the solvent fraction of the water lily root.

 4 and 5 are views showing the glucose intake activity of the water lily root extract and fractions and the separated compounds.

 4 is a diagram showing the glucose uptake activity of L6 cells, which are the murine skeletal muscle cells of the extract and fraction of the water lily root.

 5 is a diagram showing the glucose intake activity in L6 cells, which are skeletal muscle cells of the mouse of Formula 1, Formula 2, Formula 3 or Formula 4.

[Form for implementation of invention]

 Hereinafter, the present invention will be described in detail. The present invention provides a pharmaceutical composition for the prevention and treatment of metabolic diseases containing water lily root GVJ 3es tetragona) extract as an active ingredient.

In addition, the present invention provides a composition comprising a water lily root extract for use in the prevention and treatment of metabolic diseases. As used herein, the term "^^ Nymphaea tetragonaY" means all organs of natural, hybrid, or cultivar, including all roots, branches, branches, leaves, and flowers. .

The extract may be extracted with water, a solvent of C ₄ alcohol or a mixture thereof, and specifically, the alcohol may be ethanol or methanol, but is not limited thereto.

The water lily root extract ^: may be prepared by a manufacturing method comprising the following steps, but is not limited thereto:

 1) extracting by adding an extraction solvent to the water lily root;

 2) cooling the extract of step 1) and then filtering; And

 3) drying the filtered extract of step 2) under reduced pressure.

 In the above method, the water lily root of step 1) can be used without limitation, such as being grown or commercially available. The water lily root may be to use the water lily underground, but is not limited thereto.

 Extraction method of the water lily root extract may be used in the art, such as filtration, hot water extraction, immersion extraction, reflux kinkak extraction and ultrasonic extraction, may be one to five times by the hot water extraction method, More specifically, the extraction may be repeated three times, but is not limited thereto. The extraction solvent may be added 0.1 to 10 times to the dried water lily root, it is preferable to add 0.3 to 5 times. The extraction temperature may be 20 to 40, but is not limited thereto. In addition, the extraction time may be 12 to 48 hours, but is not limited thereto.

 In the above method, the decompression concentration in step 3) may be to use a vacuum decompression concentrator or a vacuum rotary evaporator, but is not limited thereto. In addition, the drying may be, but not limited to, drying under reduced pressure, vacuum drying, boiling drying, spray drying or freeze drying.

The metabolic disease consists of obesity, type 2 diabetes, dyslipidemia, insulin resistance, hepatic steatosis and non-alcoholic fatty liver. It may be selected from the group, but is not limited thereto. In addition, the present invention provides a pharmaceutical composition for preventing and treating metabolic diseases, including a fraction obtained by fractionating the water lily root extract using an organic solvent. The present invention also provides a composition comprising an organic solvent fraction of water lily root extract for use in the prevention and treatment of metabolic diseases.

 The organic solvent may be selected from the group consisting of nucleic acid, chloroform and butanol, but is not limited thereto.

 The fraction may be a nucleic acid fraction, chloroform fraction, butanol fraction or water fraction obtained by systematic fractionation of water lily root extract in the order of hexane, chloroform, butanol and water, but is not limited thereto. Preferably, the nucleic acid is n-nucleic acid.

 The metabolic disease may be selected from the group consisting of obesity, type 2 diabetes, dyslipidemia, insulin resistance, hepatic steatosis, and nonalcoholic fatty liver, but not limited thereto. In addition, the present invention provides a pharmaceutical composition for the prevention and treatment of metabolic diseases containing a polyphenol-based compound of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

<Formula 1>

 In addition, the present invention provides a pharmaceutical composition for the prevention and treatment of metabolic diseases containing a polyphenol-based compound of Formula 2 or a pharmaceutically acceptable salt thereof as an active ingredient.

 <Formula 2>

또한, 본 발명은 하기 화학식 3의 클리페놀계 화합물 또는 이의 약학적으로 용가능한 염을 유효성분으로 함유하는 대사성 .질환 예방 및 치료용 약학적 조성물을 제공한다.

The present invention also provides a pharmaceutical composition for the prevention and treatment of metabolic diseases-containing a cliphenol-based compound of Formula 3 or a pharmaceutically acceptable salt thereof as an active ingredient.

 <Formula 3>

 In addition, the present invention provides a pharmaceutical composition for the prevention and treatment of metabolic diseases containing a polyphenol-based compound of Formula 4 or a pharmaceutically acceptable salt thereof as an active ingredient.

 <Formula 4>

화학식 1，화학식 2,화학식 3또는 화학식 4의 폴리페놀계 화합물，또는 0 약학적으로 허용가능한 염을 제공한다. 상기 화학식 1， 화학식 2， 화학식 3 및 화학식 4의 화합물은 각각 수련뿌리 추출물로부터 분리한 것일 수 있으나 이에 한정하지 않으며， 다른 물질로부터 유래한 것 또는 합성한 것 모두사용가능하다. . In addition, the present invention is for use in the prevention and treatment of metabolic diseases

It provides a polyphenolic compound of Formula 1, Formula 2, Formula 3 or Formula 4, or 0 pharmaceutically acceptable salts. Compounds of Formula 1, Formula 2, Formula 3 and Formula 4 may be isolated from the water lily root extract, respectively, but are not limited thereto, and those derived from other substances or synthesized may be used.

 The metabolic disease may be selected from the group consisting of obesity, type 2 diabetes, dyslipidemia, insulin resistance, hepatic steatosis, and nonalcoholic fatty liver, but is not limited thereto. The present inventors pulverized the water lily root to prepare a water lily root extract and fractions were extracted by dipping in methanol. The extract was filtered, concentrated under reduced pressure to obtain methane. The extract was extracted with methane, chloroform, butanol, and water, and the fractions were extracted to obtain active fractions. — Inhibition of 3-phosphate acyltransferase (huraan glycerol-3-phosphate acyl transferase (hGPATl)) showed that butane was better inhibited in fractions.

 In order to separate the compound from the water lily root extract, the present inventors separated the butanes into eight fractions using reverse phase column chromatography while increasing the polarity sequentially with a methanol / water mixed solvent. (Fractions 1 to 8). Among them, the fractions with the strongest inhibitory activity were collected and the active fractions were separated by eluting with methane using Sephatex LH-20 (Sephadex LH20). Among them, fractions with strong inhibitory activity were collected, and high-performance liquid chromatography (YMC J'sphere ODS H-80 column, 250 20 讓) was carried out by pouring methanol / water mixed solvent at 5 ml / min as elution solvent. Four compounds (Formula 1, Formula 2, Formula 3, and Formula 4) were obtained.

To determine the structure of the four compounds, the present inventors have determined the properties, molecular weights, molecular formulas, mass spectrometry, ^' ¾-NMR spectra, and ¹³ C-NMR spectra of the compounds as disclosed in Hideyuki Kurihara et a. \. Biosci. Biotech. Biochem. , 57 (9): 1570-1571, 1993; Wen-Hua Zhao et al. J. Chroma togr. B., 850, 523-527, 2007; RW Owen et al. Food and Chemical Toxicology. , 41, 1727-1738, 2003], geranin,

1,2,3,4,6-penta-galloic-beta beta-glucose (1,2,3,4, 6-pent a-i> gal 1 oy 1 bet a-glucose),

 1, 2, 3, 6-tetra-0-gallo-beta-eglucose (1, 2, 3, 6-tetra-0-ga 11 oyl-beta-Dg 1 ucose), methyl gallate ). Physicochemical properties and chemical structure of the compound isolated from the water lily root are as follows.

 <Formula 1>

 Geranin

 <Formula 2>

1,2,3,4, 6-penta—galloy-beta-glucose (1,2,3,4, 6-pent a- ^ g lloyl -beta— D 一 glucose)

 <Formula 3>

 1,2 ᅳ 3，6 ᅳ tetrazagalloy-beta-glucose (1,2,3,6—tetra-galloyl-b eta—D—glucose)

<화학식 4>

<Formula 4>

 Methyl gallate

The present inventors cloned human hGPATl cDNA (NCBI accesstion No. NM ᅳ 020020) into pFastBacl vector and transfected recombinant bacmid DNA into Sf9 cells in order to isolate the mass expression of hGPATl (human GPAT1) and hGPATl enzyme source. By converting, a baculovirus containing human hGPATl 0 ^ ^' was assembled and amplified. Cells were recovered and homogenized using an ultrasonic cell crusher. Only supernatant was taken and centrifuged. The obtained precipitate was suspended in a complete sucrose solution and used as an enzyme source of mitochondrial hGPATKmitochondrial hGPATl). In addition, the precipitate obtained by centrifugation of the supernatant from which the precipitate was removed was suspended in sucrose buffer and used as a microsomal hGPATl enzyme source.

 In order to measure the inhibitory effect of human-derived hGPATl, the activity of microsomal hGPATl enzyme is lost by N ᅳ ethylmaleimide (NEM). When measuring only mitochondrial hGPATl activity, N- The experiment was carried out after the microsomal hGPATl activity was lost by pretreatment with ethylmaleimide. The activity of mitochondria hGPATl enzyme is

The radiation dose of [ ¹⁴ C] lysophosphatidic acid ([ ¹⁴ C] Lysophosphat lic acid) was measured. The activity of mitochondrial hGPATl was measured in an ice bath with 2 mM N 2ethylmaleimide for 15 minutes in the mitochondrial hGPATl enzyme source before the experiment. After deactivation of microsomal hGPATl by pretreatment, microsomal hGPATl activity was measured by pretreatment of N-ethylmaleimide (2 mM) with microsomal hGPATl enzyme source to determine the ratio of total hGPATl activity and enzymatic activity of microsomal hGPATl. The enzyme activity was measured by correcting the activity result of the microsomal hGPATl enzyme source.

Substrates used for enzyme reaction were [ ¹⁴ C] glycerides -3 ᅳ phosphate (1.8 μΜ) and palmitoleyl-CoA (100 μΜ). The reaction was stopped by water and water. The reaction product was separated into a water layer and a butanol layer by centrifugation.

A supernatant (layered butane) containing [ ¹⁴ C] lysophosphatidic acid was taken. The supernatant was shaken with the same amount of water and separated again into a water layer and a butanol layer by centrifugation. The supernatant was taken up to 600 χ to obtain the amount of radioactivity (Disintegrations per minute (DPM)) using a liquid scintillation counter (LSC). Measured. The hGPATl enzyme activity was measured by treating the water lily extract, fractions and compounds of Formula 1, Formula 2 and Formula 3 prepared in Examples 1 and 2, respectively. As a result, the water lily root extract, fractions thereof, or acyclic polyphenol-based compounds isolated therefrom showed excellent hGPATl inhibitory activity and showed hGPATl inhibitory activity in a concentration-dependent manner (see Table 1, Table 2, Figs. 1 and 2). ).

 In order to determine the effect on the synthesis of triglycerides in the cells by the compound of the present invention, HepG2 cells, which are human-derived hepatocytes, were used to measure the triglyceride biosynthesis inhibitory activity in cells against two substrates. As a result, as shown in Table 3, the water lily extract and the fractions were treated at a concentration of 50 /, respectively.

When using [ ¹⁴ C] glycerol as a substrate, each fraction inhibited the production of triglycerides in cells (see Table 3).

In order to measure the effect of increasing glucose intake in L6 cells (rat skeletal myoblast cell line), after treatment with extracts or fractions of indigenous L6 cells including inslin and the formulas 1, 2, 3 and 4 Incubated in Krebs-Singer Buffer (KB). Cells cultured in RB were treated with 2-deoxy-D-gkicose and labeled 2-deoxy-2-glucose (2-deoxy- [14C] glucose, 0.2 Ci / ml). After treatment, reaction was carried out for 10 minutes. After the reaction, the glucose intake was determined by measuring with a scintillation counter. As a result, when treated with a ½ root extract or butanol fraction significantly increased glucose uptake compared to the control (DMS0). In addition, the treatment of Formula 1, Formula 2, Formula 3, Formula 4 significantly increased glucose uptake compared to the control (DMS0)-.

In order to measure the effect of increasing the glucose uptake in the L6 cells of the fraction L6 cells (rat skeletal myoblast cell line), the water extracts of Butyl root extract or butane were treated with fractions of the water extracts and butane including insulin, and then Krebs-Ringer buffer (Krebs-Ringer Buffer, KRB). Cells cultured in KRB were labeled with 2-deoxy-D-glucose and labeled 2-deoxy-2-glucose, 2-deoxy-D- [ ¹⁴ C] glucose, 0.2 uCi. / ml) and reacted for 10 minutes. After the reaction, the glucose intake was determined by measuring with a scintillation counter. As a result, the treatment of water extract or butane significantly increased glucose uptake compared to the control group (DMS0) and was dependent on insulin.

In order to determine the oral acute toxicity of the water lily root extract of the present invention, the following experiment was performed using a mouse. The experimental animals were specific pathogen free (SPF) animals, and 7-week-old male ICR mice were divided into a control group and a test group, and the control group received only 0.5% carboxymethyl cellulose (CMC) solution. The test group was orally administered to the water lily root extract of the present invention prepared in Example 1 at a dose of 1,000 mg / kg, 500 mg / kg for 2 weeks to observe the toxicity. As a result, no animals died in all groups, and no animals with unusual behavior or toxic symptoms were found. In addition, body weight gain was observed in all groups, and abnormal lesions and abnormal findings in all organs in the thoracic cavity and abdominal cavity observed at autopsy Not found.

 Therefore, the water lily root extract, fractions thereof, and polyphenolic compounds isolated from the fractions effectively inhibit the activity of mtGPATl causing metabolic disease, inhibit the biosynthesis of intracellular triglycerides and insulin in skeletal muscle cells. Since it was confirmed that the dependent glucose uptake activity, the polyphenol-based compounds of the present invention, water extract root or fractions thereof are obesity, type 2 diabetes, dyslipidemia, insulin resistance, hepatic steatosis and non-alcoholic fatty liver, etc. It can be used as an active ingredient of a pharmaceutical composition for the prevention and treatment of metabolic diseases.

 It contains 0.1 to 99.9% by weight of the water lily root extract of the present invention or a fraction thereof based on the total weight of the composition of the present invention as an active ingredient, and may include a pharmaceutically acceptable carrier, excipient or diluent.

The compositions of the present invention may be in various 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, powders, granules, capsulants, and the like, which may contain at least one excipient such as starch, carbohydrate, sucrose or lactose ( lactose) and gelatin. In addition, the ^"lubricant such as magnesium stearate, in addition to simple excipients, thoracic, and talc are also used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. As the non-aqueous and suspending solvent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As a base of suppositories, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used. The composition of the present invention can be administered orally or parenterally, and it is preferable to select the external or intraperitoneal, rectal, intravenous, intramuscular, subcutaneous intrauterine dural or cerebrovascular injection method for parenteral administration. Use for external skin.

 The dosage of the composition of the present invention varies depending on the weight, age, sex, health status, diet, time of administration, administration method, excretion rate and severity of the disease of the patient, the daily dosage of the extract 0.01 to 1000 mg / kg based on the amount, preferably 30 to 500 mg / kg, more preferably 50 to 300 rag / kg, may be administered 1 to 6 times a day.

 The composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological reaction modifiers. The present invention also provides a method for preventing metabolic disease comprising administering to a subject a pharmaceutically effective amount of the composition according to the present invention.

 The present invention also provides a method of treating a metabolic disease comprising administering a pharmaceutically effective amount of the composition according to the present invention to a subject having metabolic disease.

 The metabolic disease may be any one selected from the group consisting of obesity, type 2 diabetes, dyslipidemia, insulin resistance, hepatic steatosis, and nonalcoholic fatty liver, but is not limited thereto.

 The pharmaceutically effective amount is 0.0001 to 100 mg / kg, 0.001 to

10 mg / kg, but is not limited thereto. Dosage may vary depending on the weight, age, sex, health status, diet, duration of administration, rate of elimination, and severity of the particular patient.

The composition can be administered orally or parenterally during clinical administration and intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, It can be administered by intrauterine epidural injection, cerebrovascular injection, or intrathoracic injection, and can be used in the form of general pharmaceutical preparations.

 The subject is a vertebrate, specifically a mammal, more specifically an experimental animal such as a rat, rabbit, guinea pig, hamster, dog, or cat, and more specifically, may be an ape-like animal such as a chimpanzee or a gorilla. have.

 In the present invention, "administration" means introducing a certain substance into a patient in any suitable way and the route of administration of the substance can be administered via any general route as long as it can reach the target tissue. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration topical, administration, intranasal administration, pulmonary administration, rectal administration, but is not limited thereto. In addition, the pharmaceutical composition may be administered by any device that allows the active substance to migrate to the target cell.

 In the present invention, "administration" means that the composition of the present invention is introduced by "systemic delivery" or "topical delivery" to pancreatic cancer cells. "Whole body delivery" refers to delivery that causes widespread biodistribution of a compound in an organism. Some administration techniques result in systemic delivery of certain compounds, and others may not. Systemic delivery means that a useful, preferably therapeutically effective amount of a compound is exposed to most of the body. Generally, in order to obtain a broad biodistribution, fast non-specific cell binding or by a fast passage of organs (liver, lung, etc.) to ensure that the compound is not rapidly degraded or eliminated before reaching the disease site far from the site of administration. Life expectancy in the blood is required. As used herein, "topical delivery" refers to the delivery of a compound directly to a target site in an organism. For example, the compound can be delivered locally by injection directly into a disease site, such as a tumor or other target site, such as a target organ, such as the pancreas.

In another aspect, the present invention provides a composition for preventing and improving metabolic diseases containing water lily extract as an active ingredient. The present invention provides a composition for preventing and improving metabolic diseases, including a fraction obtained by fractionating a water lily root extract using an organic solvent.

 The present invention also provides a composition for preventing and improving metabolic diseases, which contains a polyphenolic compound or a pharmaceutically acceptable salt thereof as an active ingredient.

 In addition, the present invention provides the use of water lily root extract for use in the manufacture of a composition for the prevention and improvement of health foods for metabolic diseases.

 In addition, the present invention provides the use of the organic solvent fraction of the water lily root extract for use in the preparation of the composition for the health food for preventing and improving metabolic diseases. In addition, the present invention provides a use of the polyphenol-based compound of Formula 1, Formula 2, Formula 3 or Formula 4, or a pharmaceutically acceptable salt thereof for use in the preparation of a health food composition for preventing and improving metabolic diseases to provide.

 The polyphenol-based compound is represented by Chemical Formula 1, Chemical Formula 2, Chemical Formula 3 and Chemical Formula

It may be selected from the group consisting of four, but is not limited thereto.

 The metabolic disease may be selected from the group consisting of obesity, type 2 diabetes mellitus, dyslipidemia, insulin resistance, hepatic steatosis and nonalcoholic fatty liver, but is not limited thereto.

In the present invention, the water lily root extract, its fractions and polyphenol compounds isolated from the fractions have been found to effectively inhibit the activity of mtGPATl causing metabolic diseases, including the polyphenolic compounds of the present invention, water lily root extract or Fractions thereof may be used as an active ingredient in the composition for the prevention and improvement of metabolic diseases such as obesity, type 2 diabetes, dyslipidemia, insulin resistance, hepatic steatosis and non-alcoholic fatty liver. The functional food of the present invention may contain various ^' flavours, natural carbohydrates, and the like as additional ingredients. Natural carbohydrates mentioned above include glucose, fructose and Disaccharides such as monosaccharides, maltose, sucrose, and polysaccharides such as dextrin, cyclodextrin, xyli, sorby, erythr, and the like. As the sweetening agent, natural sweetening agents such as tautin and stevia extract, synthetic sweetening agents such as saccharin and aspartame can be used. The ratio of natural carbohydrate and water may be selected from 0.01 to 0.04 parts by weight, specifically about 0.02 to 0.03 parts by weight, per 100 parts by weight of the health food of the present invention.

 In addition to the above, the functional food of the present invention includes various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols. And the carbonizing agent used in the carbonated beverage. In addition, the functional food of the present invention may contain a flesh for preparing natural fruit juice, fruit juice beverage and vegetable beverage. These components can be used independently or in combination. The proportion of such additives is not critical but is usually selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the health food of the present invention. Hereinafter, the contents of the present invention will be described in more detail with reference to Examples and Preparation Examples. However, the following Examples and Preparation Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Examples and Preparation Examples.

Example 1 Preparation of Water Lily Root Extract and Fractions

5 kg of dried water lily roots were pulverized and immersed in 25, and extracted twice. The extract was filtered, concentrated under reduced pressure, and about 199 _g of methane was obtained. Separating the active ^substance, was suspended in the water lily root extract methane in 199 g of distilled water to 1000 ^ nucleic acid, chloroform, butanol, water, the solvent fraction to obtain each of the fractions. As a result of measuring hGPATl enzyme inhibitory activity of the fractions, butane showed better inhibitory activity in the fractions.

Example 2 Separation of Compound from Water Lily Root Extract The butanes fraction (about 25 g) _. A total of 8 fractions were separated by reverse phase column chromatography with increasing polarity sequentially with a 20%, 30%, 40%, 50% methanol / water mixture (fractions 1 to 8). .

 Among them, fractions with the strongest inhibitory activity were collected and Sephatex LH-20 (Sephadex)

LH20): was used to elute the methane to separate the active fractions. Among them, fractions with strong inhibitory activity were collected, and high-performance liquid chromatography (YMC J ¹ sphere ODS H-80 column, 250X20 mm) was performed by flowing 90% methanol / water mixed solvent at 5 mi / min as the elution solvent. Four pure compounds (Formula 1, Formula 2, Formula 3, and Formula 4) were obtained.

Example 3 Characterization of the Compound

The compound was determined by measuring the properties, molecular weight, molecular formula, mass spectrometry, -NMR spectrum and ¹³ ONMR spectrum of the material, published by Hideyuki Rurihara et al. Biosci. Biotech. Biochew. , 57 (9): 1570-1571, 1993; Wen-Hua Zhao et al. J. Chromatogr. B., 850, 523-527, 2007; RW Owen et al. Food and Chemical Toxicology. , 41, 1727-1738, 2003], which is consistent with the data of Geranin (Formula 1), 1,2,3,4,6Kenta- ^ galoitzbeta-euglucose (1, 2,3,4,6 6 £ 1 ᅳ ^ 3110 卜 3 ᅳ glucose) (Formula 2), 1,2,3,6 ᅳ tetra-agalloy-beta-D-glucose

(l, 2,3,6-tetra-()-galloyl-beta-D-ghicose) (Formula 3) and methyl gallate (Formula 4) were identified. Physicochemical properties and chemical structure of the compound isolated from the water lily root are as follows.

[Formula 1] ΊΖΊ6Ι 'TS ^' 89T 'ΖΤ ^' 89ΐ 'Π ^' 99ΐ ^' 99 ^' 591 '9S ^' 99T ^' Τ8 9Ι ΌΖ ^' ^ 91 ^' 9 ^' ½Ι 'S ^' SH '6S ^' SH 'S2 ^' 9H 'CT ^' SH '96 ^' m ^' 06' T9 ^' m' S ^' SW '89 ^" 6εΐ Ί9Ί £ 1' Zf9 l '8S ^" 82I '99 ^" 921 H ['09 Ίΐ' 92O2T ^' 5-6ΙΤ' Z ^' Sn' T2 ^' STI' 9S ^' Sn '98 ^" ΟΤΤ ^' 99 ^' OXI '2S ^" 0II '22 ^" OTI' O ^' SOT ^' 88 ^' ZOT 'T2-96' 8S ^' 26' ΖΖ ^" Τ6 Ό Ό6 '0 ^' £ L 'ZO ^' SZ ^{'09 "2 'I9"0Z'} S6 '69 '98' 99 '06 "S9 '6Z' S9 '99 'S9'¾" S9 '9S "29'6" IS '6Γ9 ^: (() 9 '00 ¾3 ^'ζ Η1¾00Τ) Η -3 _εΐ (9

(ᅳ re ^§ ns) q £--ez ^' 9

'(OT ^ UIOJB) S'9-Τε ^' Ζ '(9υμπ9 ^[ ιι) xg-g ： ((uidd) g' αο) 'ZHW00 NH _T (5

90 ^' 16' ₊ = z / iu ： ^ y¾ ^^-isa (f

^L W ^lp D ： y \ ^ (£

 296 ： i¾ ^ ¾- (Z

(P! Ios U Ojq 9iBd) [^ ir ： (χ

SZ90TO / ZlOIM¾ / 13d 92 8 ££ S80 / eiOZ OAV [Formula 2]

 1) Appearance ： pale brown solid

 2) Molecular weight ： 940

 3) Molecular formula: C41H32O36

4) ESI-mass spectrometry ： m / z = [M + Na] ⁺ , 963

5) ¾-MR (400 MHz, CD ₃ 0D, δ (ppm)): 7.10 (2H, s, 1-Gall-H ᅳ 2, 6), 7.04 (2H, s, 2-Gall -H-2, 6 ), 6.97 (2H, s, 3-Gal 1 -H-2, 6), 6.94 (2H, s, 4-GaU ᅳ H-2, 6), 6.89 (2H, s, 6— Gall-H— 2 , 6), 6.22 (1H, doublet, J = 9.5 Hz, glu-H-1).

6) ¹³ C-NMR (100 MHz, CD ₃ 0D, δ (ppm)): 167.9 (2-Gal LC-7), 167.3 (l-Gall-C-7), 167.0 (3-G l LC-7) , 166.9 (4-Gal lC-7), 166.2 (6-G l l-7), 146.5 (l-Gall-C-3,5), 146.4 (2-Gal lC-3,5), 146.4 (3 -Gal 1— 03, 5), 146.3 (4-Gall-C-3,5), 146.3 (6-Gal lC-3,5), 140.8 (l-Gall-C-4), 140.4 (2-Gal lC-4), 140.3 (3-Gal lC-4), 140.1 (4-Gal lC-4), 140.0 (6-Gal lC-4), 121.1 (1-Gal 1-Cl), 120.4 (2-Gal 1-Cl), 120.3 (3-Gal 1-Cl), 120.2 (4-Gal 1-Cl), 119.7 (6-Gall-Cl), 110.6 (1-Gal lC-2,6), 110.4 (2- Gal lC-2,6, 3-Gal lC-2,6), 110.3 (4-Gall-C-2,6, 6— Gal l—C— 2,6), 93.8 (glu-C-1), 74.4 (ghi— C— 3), 74.1 (glu-C-5) , 72.2 (glu-C-2), 69.8 (ghi— C-4), 63.1 (glu-C-6).

[Formula 3]

 1) Appearance ： pale brown solid

 2) Molecular weight ： 788

3) Molecular Formula: C ₃₄ H _2S 0 ₂₂

4) ESI'mass spectrometry: m / z = [M + Na] ⁺ , 811

5) ¾-NMR (400MHz, CD ₃ 0D, δ (ppm)): 7.11 (2H, s, 6-Gall-H-2, 6), 7.01

(2H, s, 3-Gall-H-2, 6), 6.98 (2H, s, 1-Gall-H-2, 6), 6.90 (2H, s, 2-Gall-H-2, 6), 6.00 (1H, d 'J = 9.5 Hz, glu-H-1).

6) ¹³ C-NMR (100 MHz, CD ₃ 0D, δ (ppm)): 168.1 (6-Gal LC-7), 167.7 (3-Gall-C-7), 167.5 (2— Gal LC-7), 167.1 (l-Gall-C-7), 146.5 (1-Gal lC-3,5), 146.4 (3— Gal lC-3,5), 146.3 (6-Gal lC-3,5), 146.2 (2 -Gal lC-3,5), 121.2 (6-Gall-Cl), 120.8 (3-Gal 1-Cl), 120.5 (2-Gall'Cl), 120.4 (1-Gal 1-Cl), 110.4 (l -Gall-C-2,6, 2-Gal LC-2,6), 110.3 (3 ~ Gal LC-2,6, β-Gal LC-2,6), 91.5 (glu-C-1), 73.8 (glu-C-3), 73.4 (glu-C-5), 70.6 (glu-C-2), 68.7 (glu-C-4) 64.4 (glu-C-6 ).

[Formula 4]

 1) Appearance ： white powder

 2) Molecular weight ： 184

 3) Molecular formula: C8H805

 4) 1 H-NMR (400 MHz, CD30D, δ (ppm)): 7.41 (IH, s, H-2), 7.75 (IH, s, H-6), 3.87 (3H, s, methoxy methyl), 3.83 ( IH, s, ester methyl)

 5) IX— NMR (100 MHz, CD30D, δ (ppm)): 121.5 (01), 110.6 (02), 149.9 (C-3), 146.1 (C-4), 141.2 (05), 118.7 (06), 168.2 (07), 56.8 (methoxy methyl), 49.5 (ester methyl). Example 4 Mass Expression and Isolation of Enzyme Sources of Human-Derived hGPATl (human GPAT1)

Mitochondrial protein isolated from sf9 cells overexpressing Human GPATK hGPATl) was used as the enzyme source. hGPATl cDNA (NCBI accesstion No. リ _020918) was cloned into pFastBacl vector (Invitrogen), and the resulting recombinant bacmid DNA was transformed into Sf9 cells to assemble and amplify baculovirus containing human hGPATl cDNA. . Sf9 cells (1 × 10 6 cells / ral) were infected with 10 M () I virus and cells were harvested by centrifugation after 48 hours, homogenize buffer (250 mM sucrose, 10 niM Tris (H). 7.4) ， then 1 mM EDTA) Homogenization was performed using an ultrasonic cell crusher. Centrifugation (600 g, 15 minutes) to take only the supernatant, which was again centrifuged for 15 minutes at 8,000 g of the precipitated mitochondrial protein fraction sucrose buffer (250 mM sucrose, 10 mM Tris (pH 7.4) ), Suspended in 1 mM EDTA, 1 mM DTT) of the protein by the Bradford Method. The concentration was determined, which was stored ^at ᅳ 80 ^° C and used for the experiment.

Example 5 Measurement of Inhibitory Activity of Human-Derived hGPATl

 The activity of hGPATl enzyme was measured by the amount of radiation of [14C] lysophosphatidic acid ([l] Lysophosphaticlic acid), a reaction product produced by the mitochondrial hGPATl enzyme source. Mitochondrial hGPATl activity was compared to other isoenzymes, and 2 mM N—ethylmaleimide was added to the hGPATl enzyme source before the experiment, according to previous studies, in which hGPATl was maintained by treatment with ethylmaleimide (NEM). Pre-treatment in an ice bath for minutes. Deactivated isozyme activity other than hGPATl.

Substrates used for enzyme reaction were [ ¹⁴ C] glycerol -3-phosphate (1.8 μΜ) and palmitoyl-CoAKlOO μΜ and these substrates were the reaction solution (75 mM Tris (pH 7.5), 4 After reaction for 20 minutes at 26 ^° C. with mM MgCl ₂ , 8 mM NaF, 2 mg / i BSA), reaction was stopped by saturated butanol and water. The reaction was centrifuged to separate the water layer and the butane, and the supernatant containing the reaction product [ ¹⁴ C] lysophosphatidic acid (layer of butane) was taken up to 800 ^. The supernatant was shaken with the same amount of water, and the water layer and butane were separated into layers by centrifugation, and the supernatant was taken up to 600 ^ to obtain a liquid scintillation counter (LSC). ) Was measured. The final concentration of the water lily root extract and fractions prepared in Examples 1 and 2 was treated at a concentration of 30,100 g /, the final concentration of the compounds of Formula 1, Formula 2 and Formula 3 prepared in Example 2 100, 30 ， 10 ， 3 ， 1, hGPATl enzyme by treatment at concentration of 0.3 / g / m £ Activity was measured. The inhibition rate of hGPATl enzyme activity by the sample was calculated by the following equation.

 Inhibitory activity (%) = 1-[(T-B (C-B)] X 100

 τ ： DPM value of the test sphere in which the sample was added to the enzyme reaction solution ，

 C: DPM value of the control group without the sample in the enzyme reaction solution,

 B: DPM value of the control in which the sample was put without the enzyme source.

 . As a result, as shown in Table 1, Table 2, Figures 1 and 2, fractions of the water lily root extract or acyclic polyphenol-based compounds isolated therefrom have excellent hGPATl inhibitory activity and concentration-dependent hGPATl inhibitory activity. Indicated. In addition, IC50 values, which are concentrations showing 50% inhibitory activity against hGPATl of the compounds of Formulas 1, 2 and 3, were 16.9 ug / ml, 70.3 / g / and 59.8 ^ g / ml, respectively.

Table 1

【표 2]

[Table 2]

Example 6 Measurement of Triglyceride Biosynthesis Inhibitory Effect in HepG2 Cells

^ To investigate the effect of ¾ compounds on triglyceride synthesis in cells, hepG2 cells, which are human-derived hepatocytes, were used for two substrates. Triglyceride biosynthesis inhibitory activity in cells was measured. HepG2 cells were cultured at ATCC, with minimum essential medium (MEM; 2 mM L ᅳ glutamine; 1.5 g / L sodium bicarbonate, 0.1 mM essential amino acid nonessential amino acids). And 10% fetal bovine serum (FBS) and antibiotics (100 U / ml. Penicillin and 100 g / ml streptomycin) in earle ^ BSS medium formulated to contain 1 mM sodium pyruvate. Addition was incubated in a 37 ^° C, 5% C0 ₂ incubator. Reduction of hGPATl 'enzyme activity in the cell is cultured with the amount of triglyceride produced in HepG2 cells ^■ measurement was enzyme activity-inhibiting substance with formula (I) and formula (2) eu generation of after each addition of the compound of formula (3) triglyceride According to determine the effect of the enzyme inhibitor. _{In a} 24-well culture dish, the cells are aliquoted at a cell number of IX 10 ⁶ cells / ml per well, incubated for 24 hours, and then exchanged with DMEM (Dulbecco ^ modified Eagle ^t s medium) without FBS. 0.2 niCi

[ ¹⁴ C] acetate ([ ¹⁴ C] acetate) (Amersham) was added and reacted for 6 hours. Also used in other substrate ^[14 C] In the case of the glyceryl ^([14 C] glycerol), the reaction was carried out for 18 hours. The sample was dissolved in dimethyl sulfoxide (DMS0), and the control group in the neutron room production reaction was reacted only with dimethyl sulfoxide without adding the sample, and the production rate of triglycerides was 100. .

[ ¹⁴ C] acetate, a substrate that is not absorbed by the cells and remains in the medium to determine the amount of triglycerides produced in the finished cells

[ ¹⁴ C] Glyceol was removed, and once removed with phosphate-buffer-sa line (PBS), followed by extraction solvent (hexane: isopropanol = 3: 2, v: v). 0.5 ml was added to extract total fat, including triglycerides. After extracting twice with 0.5 ml of extraction solvent twice for 30 minutes, 1 ml of the extract was concentrated through nitrogen gas, and the total fat was removed after removing the extraction solvent _; It was dissolved in an organic solvent (chloroform: methane = 2: 1) and added to thin layer chromatography (TLC: silica gel 60F ₂₅₄ , thickness: 0.5 Pa, Merck) to develop a developing solvent (nucleic acid: diethyl ether: acetic acid = 80: 20: 1, v: v: v). Neutral on TLC fat (Rf value: 0.4), the ^[14, of triglycerides through the image analysis (FLA-7000, Fuji, Japan ) and then the photosensitive during removed after 3 hours in the film can be photosensitive to radiation of energy the TLC plate C] The amount of radioactivity was measured. The cells remaining after extraction were dissolved in 0.3 nil of 0.1 N sodium hydroxide and used to measure the protein concentration of the cells used for reaction. The amount of radioactivity of triglycerides was measured and the value obtained by dividing the protein concentration as an experimental value was used to correct the experimental error between the test groups, thereby calculating the production rate of triglycerides by extracts and fractions of the present invention.

As a result, as shown in Table 3, when the water lily extract and the fractions were treated at a concentration of 50 / g /, and [ ¹⁴ C] glycerol was used as a substrate, the production of triglycerides in the cells was 15.9% in the methanol extract, and the nucleic acid fraction was 16, The chloroform fraction 27.3%, butane fraction 27.4%, water fraction 35.6% was reduced.

 [Table 3]

<실시예 7〉 수련뿌리 추출물 또는 부탄올 분획물의 L6 세포 (rat skeletal myoblast cell line)에서의 포도당 섭취 증가 효과 측정

Example 7 Measurement of the Effect of Glucose Uptake on L6 Cells of Rat Water Extract or Butanol Fraction

L6 myoblasts were cultured in DMEM (4 mM L-glutamine, 1.5 g / L sodium bicarbonate, 4.5 g / L glucose) medium containing 10% FBS. Cell lines cultured in 24-well plates were washed twice with PBS with 80% growth on the front of the medium, followed by 0.2% FBS DMEM (4 mM L-glutamine, 1.5 g / L sodium bicarbonate, 1 g / L). Glucose) medium and incubated for 24 hours. Under these conditions, cells in myoblastes will differentiate into myotubes. Including insulin in differentiated L6 cells lily root extract and fractions 30 // g / me the formula (1), (2), the formula ^"and 3 each predetermined time (one hour to produce the compound of Formula 4 to a concentration of 30 uM, respectively, 3 hours, 6 Sichuan) and then incubated for 30 minutes in Krebs-Ringer Buffer (KB). Cells cultured in RB were labeled with 0.1 mM 2-deoxy-2 ᅳ glucose (2-deoxy glucose glucose).

_{After 2} _ deoxy ᅳ ₂ ᅳ glucose ₍₂ ᅳ _deoxy _ _D _ [ _{14 C} ] _gIucose , 0.2 _uCi / iTil) was reacted for 10 minutes. After the reaction, the KRB buffer was removed, washed three times with KRB buffer, and the cells were lysed with 1 M sodium hydroxide (NaOH). Cells lysed in 1M sodium hydroxide were measured by a scintillation counter to determine the degree of glucose intake.

 As a result, when the fractions of water lily root extract and butane were treated for 6 hours at a concentration of 30 / ᅳ, glucose intake was significantly increased compared to the control group (DMS0). In addition, when the formula (1), (2), (3) and (4) were treated for 6 hours at a concentration of 30 uM, glucose intake was significantly increased compared to the control group (DMS0).

Example 8 Acute Toxicity Test of Water Lily Root Extract

In order to determine the oral acute toxicity of the water lily root extract of the present invention, the following experiment was performed using a mouse. The experimental animals were SPF-specific animals and were introduced to 7-week-old male ICR mice (Korea Biolink, Chungbuk Negative), followed by a 1-week acclimatization period. At the age of five, five animals were assigned to each group and used in the experiment. The control group was administered only 0.5% carboxymethyl cellulose (CMC) solution, the test group was prepared in Example 1. After the oral administration of the water lily root extract of the present invention at a dose of 1,000 mg / kg, 500 rag / kg, respectively, the toxicity was observed for 2 weeks.

 As a result, no animals died in all groups, and no animals with unusual behavior or toxic symptoms were found. In addition, body weight gain was observed in all groups, and no abnormal lesions or abnormalities were found in all the organs in the thoracic and abdominal cavity that were observed at necropsy. Therefore, water extracts were 1 련 000 mg / kg and 500 mg, respectively. It was found that there was no toxicity following a single oral dose of / kg.

Preparation Example 1 Preparation of Powder

 Water lily root extract of Example 1 0.1 g Lactose 1.5 g Talc 0.5 g The above ingredients were mixed and filled in an airtight cloth to prepare a powder.

Preparation Example 2 Preparation of Tablet

Water lily root fraction of Example 1 0.1 g Lactose 7.9 g Crystalline cellulose 1.5 g Magnesium stearate 0.5 g Direct tableting method after mixing the above ingredients

Prepared.

Preparation Example 3 Preparation of Capsule

0.1 g of a compound of Formula 1 of <Example 2> Corn starch

Carboxy Cellulose

제조방법에 따라 경질 캡술에 충전하여 캡술제를 제조하였다. After mixing the above ingredients to prepare a powder, the powder through

According to the manufacturing method was filled in hard capsule to prepare a capsule.

Production Example 4 Preparation of Injection

 0.1 g of the compound of Formula 2 of <Example 2> sterile distilled water dose pH adjuster dose for injection according to the conventional method for preparing an injection was prepared in the above component content (2 iii ^) per ampule.

Production Example 5 Preparation of Liquid

10 g manny of 0.1 g isomerized sugar of <Formula 3> of <3> 5 ^' g purified water appropriate amount according to the conventional method for preparing a liquid, each of the components were dissolved in purified water, and a suitable amount of lemon flavor was added to the above components Was mixed. Then, purified water was added thereto to adjust the total amount to 100, and then layered into brown bottles to sterilize to prepare a liquid.

Production Example 6 Production of Health Foods

 100 mg of the compound of formula 4 of <Example 2>

Vitamin Complex Proper

Vitamin A Acetate 70 Mg

Vitamin E 1.0 rag

0.13 mg of vitamin Vitamin B2 0.15 mg

Vitamin B6 0.5 rag

Vitamin B12 0.2 i

Vitamin C, 10 mg

Biotin 10 μ

Nicotinamide 1.7 mg

Folic acid 50 rag

Calcium Pantothenate 0.5 mg

Mineral mixture o

Ferrous Sulfate 1.75 rag

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium phosphate mono 15 rag

Dicalcium Phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 rag

Magnesium chloride 24.8 rag

The composition ratio of the vitamin and mineral mixtures is a relatively suitable composition for a healthy food in a preferred embodiment, but may be modified arbitrarily, and the mixture of the above ingredients in accordance with the usual health food manufacturing method The granules may be prepared and used for preparing a health food composition according to a conventional method.

Production Example 6 Preparation of Healthy Drinks

 Water lily root extract of <Example 1>

Citric acid

Gorigo

Plum concentrate Taurine 1 g purified water was added to mix the above ingredients according to the general method of preparing a healthy beverage in 900 ml ^' , then stirred and heated at 85 for about 1 hour, the resulting solution was filtered and obtained in a sterile 21 container and sealed. After sterilization and refrigerated it was used to prepare a healthy beverage composition.

Although the composition ratio is a relatively suitable composition for a preferred beverage and beverage in a preferred embodiment, the compounding ratio may be arbitrarily modified according to the demand hierarchy, demand country, usage, regional and ethnic preferences.

Claims

[Range of request] 【Claim 1】. , ^■ Pharmaceutical composition for the prevention and treatment of metabolic diseases containing water lily root GVj ^ ¾ ea tetragona) extract as an active ingredient. [Claim 2] According to claim 1, wherein the extract is water, to a pharmaceutical composition for the prevention and treatment of metabolic diseases, characterized in that the extraction of the alcohol of C ₄ or a mixture thereof. [Claim 3] The pharmaceutical composition of claim 2, wherein the alcohol is ethane or methanol. [Claim 4] A pharmaceutical composition for preventing and treating metabolic diseases, comprising a fraction obtained by distilling a water lily root extract using an organic solvent. [Claim 5] The pharmaceutical composition for metabolic disease prevention and treatment according to claim 4, wherein the organic solvent is selected from the group consisting of nucleic acid, chloroform and butane. [Bill 6] The method of claim 4, wherein the fraction is a nucleic acid fraction, chloroform fraction, butanol fraction or water fraction obtained by systematically fractionating the water lily root extract in the order of nucleic acid, chloroform, butane and water. Composition. [Claim 7] Polyphenol-based compound of Formula 1 or a pharmaceutically acceptable

Pharmaceutical composition for preventing and treating metabolic diseases containing as an active ingredient: <Formula 1>

[Claim 8] A pharmaceutical composition for preventing and treating metabolic diseases comprising the polyphenol-based compound of Formula 2 or a pharmaceutically acceptable salt thereof as an active ingredient: <Formula 2>

[Claim 9] To formula (3) of the polyphenol-based compound or a pharmaceutically acceptable salt of metabolic disorders, which contains as an active ingredient for prevention and therapeutic pharmaceutical composition thereof: ^"

 [Claim 10]  A pharmaceutical composition for preventing and treating metabolic diseases comprising the polyphenol-based compound of Formula 4 or a pharmaceutically acceptable salt thereof as an active ingredient:  <Formula 4>

 [Claim 11]  The method according to any one of claims 7 to 10, wherein the compound of Formula 1, Formula 2, Formula 3 or Formula 4 is isolated from the water lily root extract. A pharmaceutical composition for preventing and treating metabolic diseases. [Claim 12] The method of claim 1, wherein the metabolic disease is obesity, crab type diabetes, dyslipidemia, insulin resistance, hepatic steatosis and nonalcoholic Pharmaceutical composition for the prevention and treatment of metabolic diseases, characterized in that it is selected from the group consisting of fatty liver. [Claim 13] Water lily root extract, fractions thereof or polyphenol-based compounds isolated therefrom as an active ingredient for metabolic disease prevention and improvement for health food composition. [Claim 14] The method of claim 13, wherein the polyphenol-based compound is a composition for preventing and improving metabolic diseases, characterized in that the selected from the group consisting of Formula 1, Formula 2, Formula 3 and Formula 4: <Formula 1>

<Formula 2>

[Claim 15] 15. The method according to claim 13 or 14, wherein the metabolic disease is selected from the group consisting of obesity, type 2 diabetes, dyslipidemia, insulin resistant hepatic steatosis, and nonalcoholic fatty liver. Composition for health food for preventing and improving metabolic diseases. [Claim 16] A method of preventing metabolic disease comprising administering to a subject a pharmaceutically effective amount of water extract or fractions thereof. [Claim 17] A method of treating metabolic disease comprising administering a pharmaceutically effective amount of a water lily root extract or a fraction thereof to a subject with metabolic disease. [Claim 18] A method of preventing metabolic disease comprising administering to a subject a pharmaceutically effective amount of the polyphenolic compound of Formula 1, Formula 2, Formula 3 or Formula 4 or a pharmaceutically acceptable salt thereof. [Claim 19] A method of treating a metabolic disease comprising administering a pharmaceutically effective amount of the polyphenolic compound of Formula 1, Formula 2, Formula 3 or Formula 4 or a pharmaceutically acceptable salt thereof to a subject having metabolic disease. [Claim 20] A composition comprising a water lily extract or an organic solvent fraction thereof for use in the prevention, amelioration or treatment of metabolic diseases. [Claim 21] A polyphenolic compound of Formula 1, Formula 2, Formula 3 or Formula 4, or a pharmaceutically acceptable salt thereof, or a composition comprising the same for use in preventing, ameliorating or treating a metabolic disease.