KR20130064034A - Pharmaceutical composition containing nymphaea tetragona extract, fractions thereof or isolated polyphenolic compounds for prevention or treatment of metabolic disease - Google Patents

Abstract

PURPOSE: A Nymphaea tetragona extract, a fraction thereof, or a polyphenol-based compound isolated from the fraction is provided to effectively suppress mtGPAT1 activity which causes metabolic diseases, to suppress biosynthesis of neutral fat in a cell, and to increase intake of insulin-dependent glucose in a skeletal muscle cell. CONSTITUTION: A pharmaceutical composition for preventing and treating metabolic diseases contains a Nymphaea tetragona extract as an active ingredient. The extract is prepared using water, C1-C4 alcohols, or a mixture thereof. The pharmaceutical composition contains a fraction which is prepared from the extract using an organic solvent. The organic solvent is selected from the group consisting of hexane, chloroform, and butanol. The pharmaceutical composition also contains a polyphenol-based compound of chemical formula 1 or pharmaceutically acceptable salt thereof as an active ingredient.

Description

Pharmaceutical composition containing Nymphaea tetragona extract, fractions or or isolated polyphenolic compounds for prevention or treatment of metabolic disease}

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

Biosynthesis of triglycerides is a multistage reaction, the first of which is catalyzed by glycerol-3-phosphate acyltransferase (GPAT), and the sn − of glycerol-3-phosphate Transfers fatty acyl-CoA, a form of acyl-coenzyme A at position 1, to catalyze an ester reaction that produces lysophosphatidic acid (LPA) and to glycerol-3 It is known as a rate-limiting step that regulates the rate of the glycerol-3-phosphate pathway (Bell et al, Annu Rev Biochem 49: 459-487, 1980). The final step is the biosynthesis of triglycerides by diacylglycerol acyltransferase ("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 including NEM, and mtGPAT1 present in the mitochondrial outer membrane (MOM). mtGPAT2 is resistant to NEM and has the characteristic of favoring saturated fatty acyl-CoA as substrate (Gonzanlez-Baro et al. Biochim. Biophys. acta, 1771: 830-838, 2007).

Although mtGPAT accounts for about 10% of total GPAT activity in most tissues of the human body, up to 50% of total GPAT activity is found in liver tissues, and it is known to be high in adipose tissues (Bell et. al, Annu Rev Biochem 49: 459-487, 1980; Lewin et al. Arch. Biochem. Biophys. 396: 119-127, 2001). This suggests that mtGPAT may be a useful target for fatty liver formation, obesity and insulin resistance therapy.

 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, mRNA expression of mtGPAT is increased and, as a result, mtGPAT activity is increased, and similarly, it is left without movement for about 10 hours after continuous exercise. In the mice group, the activity of mtGPAT was steadily increased compared to the mice in the group without exercise at all, and as a result, the synthesis of triglyceride in blood was significantly increased (KuMp et al. 2006).

Selective overexpression of mtGPAT in rats in rats increased lipid biosynthesis in liver, significantly reduced fatty acid oxidation, increased fatty liver, dyslipidemia and insulin resistance compared to controls. 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, because obesity patients have a significantly higher incidence of fatty liver, 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, ingesting high-fat, high-sugar diets to induce obesity in mtGPAT ^- deficient (mtGPAT ^- deficient) mice resulted in CPT-1 and β-oxidation in contrast to the synthesis of triglycerides. In addition, mtGPAT-deficient (mtGPAT ^-/- ) mice had a 37% decrease in triglyceride content and a 15% decrease in blood compared to controls, and very low density lipoprotein (VLDL). Secretion was reduced by 30%, and as a result, mtGPAT-/-was observed to increase insulin sensitivity after a decrease in body weight and fat mass (Hammond et al, Mol Cell Biol 22: 8024-8214, 2002; Hammond et al. J Biol Chem 280: 25629-25636, 2005).

Therefore, GPAT inhibition may be an action point to inhibit intracellular accumulation of triglycerides, and the development of small molecule 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 derivatives (Eydysh et al. Bioorg. Med. Chem. 2010, 18: 6470-6479), 2- (nonylsulfonamido) and benzoic acid derivatives ( Eydysh et al. J. Med. Chem. 2009, 52: 3317-3327), but IC ₅₀ values between 25 μM and 350 μM showed weak inhibitory activity as enzyme inhibitors. The enzyme source used is interpreted as the result of the mitochondrial fraction in rats, 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 suppressing and treating various viral infections is known (WO 2011/019498).

On the other hand, Nymphaea tetragona ) is a plant belonging to the water lily family, which is distributed in Korea, Japan, China, India, and eastern Siberia, and many sea stalks are aquatic plants, and many petioles grow on the short and short stems of flowers. 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 folklore, flowers are used as drugs for hemostasis, tonic, insomnia, etc.

Geraniin, a polyphenolic compound, and 1,2,3,4,6-penta-O-galoy-beta-D-glucose (1,2,3,4,6-penta-O) -galloyl-beta-D-glucose (PGG) is known and is known to inhibit radiation induced apoptosis (Kang et al. Cell Biol. Toxicol. 2011, 27: 83-94; Biol. Pharm Bull. 2010, 33: 1122-1127).

The present inventors effectively inhibited the enzyme activity of mtGPAT1 by inhibiting the activity of mtGPAT1 by extracting water extracts, fractions thereof, and polyphenol-based compounds isolated therefrom in the search for an active substance that inhibits mtGPAT1 from natural products. By confirming that glucose uptake in skeletal muscle cells is increased, the present invention is confirmed to be effective in preventing and treating metabolic diseases such as obesity, type 2 diabetes, dyslipidemia, insulin resistance, hepatic steatosis and nonalcoholic fatty liver. The invention was completed.

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

Another object of the present invention is to provide a pharmaceutical composition for preventing and treating metabolic diseases, including a fraction obtained by dividing the water lily root extract with an organic solvent.

In addition, another object of the present invention is a pharmaceutical composition for the prevention and treatment of metabolic diseases containing 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 2>

;

;

<Formula 3>

; 및

; And

&Lt; Formula 4 >

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

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

The present invention also provides a pharmaceutical composition for the prevention and treatment of metabolic diseases, comprising a fraction obtained by fractionating the water lily root extract using an organic solvent.

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

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 comprising the polyphenolic compound of Formula 1, Formula 2, Formula 3 or Formula 4, or a pharmaceutically acceptable salt thereof as an active ingredient. do.

Water lily root extract, fractions thereof or polyphenolic compounds isolated from the fractions according to the present invention effectively inhibit the activity of mtGPAT1 causing metabolic disease, inhibit the biosynthesis of triglycerides in the cell and insulin-dependent glucose uptake in skeletal muscle cells Since it has been confirmed that the present invention exhibits augmenting activity, the extracts containing the polyphenol-based compounds of the present invention or water extracts thereof, or fractions thereof include obesity, type 2 diabetes, dyslipidemia, insulin resistance, hepatic steatosis and nonalcoholic fatty liver, etc. It can be usefully used for the prevention and treatment of metabolic diseases.

1 is a diagram showing the inhibitory activity of hGPAT1 of the water extract methanol extract, butanol fraction and the compound isolated from the fraction.
Figure 1a is a diagram showing the inhibitory activity of hGPAT1 of the water extract methanol extract and solvent fractions.
Figure 1b is a diagram showing the hGPAT1 inhibitory activity of the formula (1), (2), (3) or (4).
2 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.
Figure 3 is a diagram showing the glucose intake activity of the water lily root extract and fractions and isolated compounds.
Figure 3a is a diagram showing the glucose intake activity of L6 cells, the rat skeletal muscle cells of the methanol extract and fraction of the water lily root.
Figure 3b is a diagram showing the glucose intake activity in L6 cells, which are skeletal muscle cells of the formula (1), (2), (3) or (4).

Hereinafter, the present invention will be described in detail.

The present invention is a water lily root ( Nymphaea It provides a pharmaceutical composition for the prevention and treatment of metabolic diseases containing tetragona ) extract as an active ingredient.

The term used in the present invention, “ Nymphaea tetragona ) ”is meant to include all organs of natural, hybrid or variegated water lilies, eg roots, branches, stems, leaves, flowers, but specifically the subterranean water lilies.

The extract may be extracted with water, a solvent of C ₁ to C ₄ 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 including 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 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 cultivated 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 a conventional method in the art, such as filtration, hot water extraction, immersion extraction, reflux cooling 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. The drying may be, but not limited to, vacuum drying, vacuum drying, boiling, spray drying or lyophilization.

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 invention also provides a pharmaceutical composition for the prevention and treatment of metabolic diseases, comprising a fraction obtained by fractionating the water lily root extract using an organic solvent.

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

The fraction may be hexane 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. It is preferable that the hexane is n-hexane.

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.

In another aspect, the present invention provides a pharmaceutical composition for preventing and treating metabolic diseases containing a polyphenol-based compound of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

&Lt; Formula 1 >

In another aspect, the present invention provides a pharmaceutical composition for preventing and treating metabolic diseases comprising a polyphenol-based compound of Formula 2 or a pharmaceutically acceptable salt thereof as an active ingredient.

<Formula 2>

In another aspect, the present invention provides a pharmaceutical composition for preventing and treating metabolic diseases containing a polyphenol-based compound of Formula 3 or a pharmaceutically acceptable salt thereof as an active ingredient.

<Formula 3>

In another aspect, the present invention provides a pharmaceutical composition for preventing and treating metabolic diseases comprising a polyphenol-based compound of Formula 4 or a pharmaceutically acceptable salt thereof as an active ingredient.

&Lt; Formula 4 >

The compounds of Formula 1, Formula 2, Formula 3, and Formula 4 may be separated from the water lily root extract, respectively, but are not limited thereto. Any one derived from another substance 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 and concentrated under reduced pressure to obtain a methanol extract. To isolate the active substance, the water extract methanol extract was solvent fractionated with hexane, chloroform, butanol, and water to obtain respective fractions, and the human glycerol-3 of the fractions. -Phosphate acyltransferase (human glycerol-3-phosphate acyltransferase, hGPAT1) inhibition activity was measured and confirmed that the superior inhibitory activity in the butanol fraction.

In order to separate the compound from the water lily root extract, the inventors separated the butanol fraction into a total of eight fractions using reverse phase column chromatography with increasing polarity sequentially with a methanol / water mixed solvent ( Fraction 1 to fraction 8). Among them, the fractions with the strongest inhibitory activity were collected and the active fractions were separated by eluting with methanol using Sephadex LH-20. Among them, fractions with strong inhibitory activity were collected, and high-performance liquid chromatography (YMC J'sphere ODS H-80 column, 250 × 20 mm) was performed by flowing methanol / water mixed solvent at 5 ml / min as elution solvent. Four pure compounds (Formula 1, Formula 2, Formula 3, and Formula 4) were obtained.

To determine the structure of the four compounds, the present inventors measured the properties, molecular weight, molecular formula, mass spectrometry, ¹ H-NMR spectrum, and ¹³ C-NMR spectrum of the compounds, published in Hideyuki Kurihara et al. . Biosci . Biotech . Biochem . 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], and geranin, 1,2,3,4,6-penta- O -galoi-beta-D-glucose (1,2, 3,4,6-penta- O- galloyl-beta-D-glucose), 1,2,3,6-tetra- O- gallo-beta-D-glucose (1,2,3,6-tetra- O-galloyl-beta-D-glucose) and methyl gallate were identified. Physicochemical properties and chemical structure of the compound isolated from the water lily root are as follows.

&Lt; Formula 1 >

Geranin

<Formula 2>

1,2,3,4,6-penta- O -galoi-beta-D-glucose (1,2,3,4,6-penta- O- galloyl-beta-D-glucose)

<Formula 3>

1,2,3,6-tetra-O-gallo-beta-D-glucose (1,2,3,6-tetra-O-galloyl-beta-D-glucose)

&Lt; Formula 4 >

Methyl gallate

The present inventors cloned human hGPAT1 cDNA (NCBI accesstion No. NM_020918) into pFastBac1 vector and transformed recombinant bacmid DNA into Sf9 cells in order to isolate the mass expression of human GPAT1 and human GPAT1 enzyme source. A baculovirus containing human hGPAT1 cDNA 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 sucrose buffer and used as an enzyme source of mitochondrial hGPAT1. 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 hGPAT1 enzyme source.

In order to measure the inhibitory effect of human-derived hGPAT1, the activity of microsomal hGPAT1 enzyme is lost by N-ethylmaleimide (NEM), and when measuring only mitochondrial hGPAT1 activity, Experiment was performed after the microsomal hGPAT1 activity was lost by pretreatment with ethylmaleimide. The activity of mitochondrial hGPAT1 enzyme was measured by the amount of radiation of the resulting reactant [ ¹⁴ C] lysophosphatidic acid ([ ¹⁴ C] Lysophosphatidic acid). The activity of mitochondrial hGPAT1 was measured after deactivation of microsomal hGPAT1 by pretreatment with 2 mM N-ethylmaleimide for 15 minutes in an ice bath to mitochondrial hGPAT1 enzyme source before the experiment. N-ethylmaleimide (2 mM) was pretreated with the microsomal hGPAT1 enzyme source to determine the enzyme activity ratio of the microsomal hGPAT1 enzyme source by measuring the ratio of total hGPAT1 activity and enzyme activity of the microsomal hGPAT1 enzyme.

Substrates used for the enzymatic reaction were [ ¹⁴ C] glycerol-3-phosphate (1.8 μM) and palmitoyl-CoA (100 μM). These substrates were reacted with saturated butanol after reaction in the reaction solution. The reaction was stopped by water. The reaction was separated into a water layer and a butanol layer by centrifugation, and a supernatant (butanol layer) containing the reaction product [ 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. 600 μl of the supernatant was taken and a liquid scintillation counter (LSC) was used to determine the amount of radioactivity (Disintegrations per minute (DPM)). Measured. HGPAT1 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 hGPAT1 inhibitory activity and showed hGPAT1 inhibitory activity in a concentration-dependent manner (see Table 1, Table 2, FIGS. 1A and 1B). ).

In order to examine 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, each of the fractions inhibited the production of triglycerides in the cells when the water extract and fractions were treated at a concentration of 50 µg / ml and [ ¹⁴ C] glycerol was used as a substrate (see Table 3). ).

In order to measure the effect of increasing glucose uptake in L6 cells (rat skeletal myoblast cell line), differentiated L6 cells were treated with water extracts or fractions including insulin and Formula 1, Formula 2, Formula 3, Formula 4 and then Krebs. -Incubated in Krings-Ringer Buffer (KRB). 2-deoxy-D-glucose and labeled 2-deoxy-D- [14C] glucose, 0.2 μCi / ml, in cells cultured in KRB ) And reacted for 10 minutes. After the reaction, the glucose intake was determined by measuring with a scintillation counter. As a result, when the water lily root extract or butanol fraction was treated significantly increased glucose intake compared to the control (DMSO). In addition, when treated with Formula 1, Formula 2, Formula 3, Formula 4 significantly increased glucose intake compared to the control (DMSO).

In order to measure the effect of increasing the glucose uptake in L6 cells (rat skeletal myoblast cell line) of water extract or butanol fraction, water-derived root extracts including insulin and butanol fraction were treated with Krebs-Ringer buffer (Krebs). -Ringer Buffer, KRB). 2-deoxy-D-glucose and labeled 2-deoxy-D- [ ¹⁴ C] glucose, 0.2 μCi / ml) was treated 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 lily root extract or butanol fraction significantly increased glucose uptake compared to the control (DMSO) and appeared to be insulin dependent.

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 7-week-old male ICR mice were divided into a control group and a test group. The control group received only 0.5% carboxymethyl cellulose (CMC) solution. In the oral administration of the water lily root extract of the present invention prepared in Example 1 at a dose of 1,000 mg / kg, 500 mg / kg, respectively, was observed for 2 weeks 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 no abnormal lesions or abnormalities were found in all organs in the thoracic and abdominal cavity that were observed at necropsy.

Therefore, the water lily root extract, fractions thereof and polyphenolic compounds isolated from the fractions effectively inhibit the activity of mtGPAT1 causing metabolic disease, inhibit the biosynthesis of triglycerides in the cell and insulin-dependent in skeletal muscle cells. Since it was confirmed that the glucose intake increasing activity, including the polyphenolic compounds of the present invention, the water extract root or fractions thereof include obesity, type 2 diabetes, dyslipidemia, insulin resistance, hepatic steatosis and non-alcoholic fatty liver. 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, pills, powders, granules, capsules, and the like, which form at least one excipient such as starch, calcium carbonate, sucrose or lactose (at least one compound). lactose) and gelatin. In addition to simple excipients, lubricants such as magnesium stearate, talc and the like are also used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, and syrups, and various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin, may be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The composition of the present invention may be administered orally or parenterally, and it is preferable to select externally or intraperitoneally, rectally, intravenously, intramuscularly, subcutaneously, intrauterine epidural or cerebrovascular injection methods for parenteral administration. For skin use externally.

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 is the amount of water extract root extract 0.01 to 1000 mg / kg, preferably 30 to 500 mg / kg, more preferably 50 to 300 mg / kg, and may be administered 1 to 6 times per day.

The composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.

The present invention also provides a method of preventing metabolic diseases comprising administering to a subject a pharmaceutically effective amount of the composition according to the 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. The dose may vary depending on the weight, age, sex, health condition, diet, administration period, method of administration, rate of elimination, severity of disease, and the like of a particular patient.

The composition can be administered orally or parenterally during clinical administration and intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine dural injection, cerebrovascular injection or intrathoracic injection during parenteral administration. And can be used in the form of general pharmaceutical formulations.

The subject is a vertebrate, specifically a mammal, more specifically an experimental animal such as a rat, rabbit, guinea pig, hamster, dog, 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, nasal 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" into pancreatic cancer cells. "Whole body delivery" refers to delivery that causes widespread biodistribution of a compound in an organism. Some dosing techniques cause systemic delivery of certain compounds, others may not. Systemic delivery means that a useful, preferably therapeutically effective amount of a compound is exposed to most of the body. In general, in order to obtain a broad biodistribution, fast nonspecific cell binding or by fast disassembly or removal of the compound (e.g., the first passage organ (liver, lung, etc.) 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.

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, comprising a polyphenolic compound or a pharmaceutically acceptable salt thereof as an active ingredient.

The polyphenol-based compound may be selected from the group consisting of Formula 1, Formula 2, Formula 3, and Formula 4, but is not limited thereto.

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.

In the present invention, the water lily root extract, fractions thereof, and polyphenol-based compounds separated from the fractions have been found to effectively inhibit the activity of mtGPAT1 causing metabolic diseases, and thus, the water lily root extract or the polyphenol-based compounds of the present invention. Its fraction may be used as an active ingredient of 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 flavors, natural carbohydrates, and the like as additional ingredients. Such natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate can 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, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols. And carbonation agents used in carbonated beverages. 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 may be used independently or in combination. The ratio of such additives is not critical but is generally 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> water lily root extract and Fraction  Produce

5 kg of dried water lily roots were ground and immersed in 25 L of methanol and extracted twice. The extract was filtered, concentrated under reduced pressure, and about 199 g of methanol extract was obtained. To isolate the active substance, 199 g of the methanol extract of water lily root was suspended in 1000 ml of distilled water, and then solvent fractioned with hexane, chloroform, butanol, and water to obtain respective fractions. As a result of measuring hGPAT1 enzyme inhibitory activity of the fractions, butanol fraction showed better inhibitory activity.

< Example  2> Isolation of Compound from Water Lily Root Extract

The butanol fraction (about 25 g) was added to a total of eight fractions using reverse phase column chromatography, with increasing polarity sequentially with 20%, 30%, 40%, 50% methanol / water mixed solvent. Separated (fraction 1 to fraction 8).

Among them, the fractions with the strongest inhibitory activity were collected and the active fractions were separated by eluting with methanol using Sephadex LH-20. Among them, fractions with strong inhibitory activity were collected and high-performance liquid chromatography (YMC J'sphere ODS H-80 column, 250 × 20 mm) was run with a 90% methanol / water mixed solvent at 5 ml / min as the elution solvent. Finally, 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, ¹ H-NMR spectrum and ¹³ C-NMR spectrum of the material, as reported by Hideyuki Kurihara et al. Biosci . Biotech. Biochem . 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], Geranin (Formula 1), 1,2,3,4,6-Penta- O -galloy-beta-D-glucose ( 1,2,3,4,6-penta- O- galloyl-beta-D-glucose (Formula 2), 1,2,3,6-tetra- O- galloy-beta-D-glucose (1, 2,3,6-tetra-O-galloyl-beta-D-glucose (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.

1) Appearance: pale brown solid

2) Molecular Weight: 952

3) Molecular formula: C ₄₁ H ₂₈ O ₂₇

4) ESI-mass spectrometry: m / z = [M + Na] ⁺ , 975.06

5) ¹ H-NMR (400 MHz, CD ₃ OD, δ (ppm)): 5.21 (methine), 7.31-6.57 (aromatic), 6.29-4.35 (sugar)

6) ¹³ C-NMR (100 MHz, CD ₃ OD, δ (ppm)): 46.19, 51.97, 62.35, 63.25, 63.66, 63.79, 65.90, 66.86, 69.93, 70.51, 72.60, 73.07, 73.30, 90.70, 91.77, 92.38 , 96.21, 107.88, 108.04, 110.22, 110.52, 110.66, 110.86, 113.36, 115.21, 115.77, 119.57, 120.26, 124.60, 124.78, 125.66, 128.58, 136.42, 137.67, 139.68, 143.42, 144.51, 144.145, 144.90, 144.90 , 145.39, 145.73, 145.92, 154.57, 164.70, 164.81, 165.36, 165.56, 166.11, 168.17, 168.31, 191.71, 194.43.

1) Appearance: pale brown solid

2) Molecular Weight: 940

3) Molecular formula: C ₄₁ H ₃₂ O ₃₆

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

5) ¹ H-NMR (400 MHz, CD ₃ OD, δ (ppm)): 7.10 (2H, s, 1-Gall-H-2, 6), 7.04 (2H, s, 2-Gall-H-2, 6), 6.97 (2H, s, 3-Gall-H-2, 6), 6.94 (2H, s, 4-Gall-H-2, 6), 6.89 (2H, s, 6-Gall-H-2 , 6), 6.22 (1H, d, J = 9.5 Hz, glu-H-1).

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

1) Appearance: pale brown solid

2) Molecular Weight: 788

3) Molecular formula: C ₃₄ H ₂₈ O ₂₂

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

5) ¹ H-NMR (400 MHz, CD ₃ OD, 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 OD, δ (ppm)): 168.1 (6-Gall-C-7), 167.7 (3-Gall-C-7), 167.5 (2-Gall-C-7 ), 167.1 (1-Gall-C-7), 146.5 (1-Gall-C-3,5), 146.4 (3-Gall-C-3,5), 146.3 (6-Gall-C-3,5 ), 146.2 (2-Gall-C-3,5), 121.2 (6-Gall-C-1), 120.8 (3-Gall-C-1), 120.5 (2-Gall-C-1), 120.4 ( 1-Gall-C-1), 110.4 (1-Gall-C-2,6, 2-Gall-C-2,6), 110.3 (3-Gall-C-2,6, 6-Gall-C- 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).

1) Appearance: white powder

2) Molecular Weight: 184

3) Molecular formula: C8H8O5

4) 1H-NMR (400 MHz, CD3OD, δ (ppm)): 7.41 (1H, s, H-2), 7.75 (1H, s, H-6), 3.87 (3H, s, methoxy methyl), 3.83 ( 1H, s, ester methyl)

5) 13C-NMR (100 MHz, CD3OD, δ (ppm)): 121.5 (C-1), 110.6 (C-2), 149.9 (C-3), 146.1 (C-4), 141.2 (C-5) , 118.7 (C-6), 168.2 (C-7), 56.8 (methoxy methyl), 49.5 (ester methyl).

< Example  4> Human body hGPAT1  ( human GPAT1 Mass expression and Enzyme  detach

Mitochondrial protein isolated from sf9 cells overexpressing Human GPAT1 (hGPAT1) was used as an enzyme source. hGPAT1 cDNA (NCBI accesstion No. NM_020918) was cloned into pFastBac1 vector (Invitrogen), and the resulting recombinant bacmid DNA was transformed into Sf9 cells to assemble and amplify baculovirus containing human hGPAT1 cDNA. Sf9 cells (1 × 10 6 cells / ml) were infected with 10 MOI of virus and 48 hours later cells were harvested by centrifugation, homogenize buffer (250 mM sucrose, 10 mM Tris, pH 7.4). ), 1 mM EDTA) was added and homogenized using an ultrasonic cell crusher. Centrifugation (600 g, 15 minutes) to take only the supernatant, which was again centrifuged at 8,000 g for 15 minutes to obtain the precipitated mitochondrial protein fraction sucrose buffer (250 mM sucrose, 10 mM Tris (pH 7.4), 1 mM EDTA, 1 mM DTT) suspended the protein concentration by the Bradford method (Bradford Method), which was stored in -80 ℃ used for the experiment.

< Example  5> Human body hGPAT1 Activity inhibition effect

The activity of the hGPAT1 enzyme was measured by the radiation amount of [14C] lysophosphatidic acid ([14C] Lysophosphatidic acid), a reaction produced by the mitochondrial hGPAT1 enzyme source. Mitochondrial hGPAT1 activity compared to other isoenzymes, hGPAT1 is maintained by the treatment of ethylmaleimide (NEM), according to the previous study, 2 mM N-ethylmaleimide was added to the hGPAT1 enzyme source before the experiment. Pretreatment in an ice bath for minutes was followed by inactivation of isoenzymes other than hGPAT1.

Substrates used for the enzymatic reaction were [ ¹⁴ C] glycerol-3-phosphate (1.8 μM) and palmitoyl-CoA (100 μM), and these substrates were the reaction solution (75 mM Tris (pH 7.5)). , 4 mM MgCl ₂ , 8 mM NaF, 2 mg / ml BSA) for 20 minutes at 26 ° C., and then the reaction was stopped by saturated butanol and water. The reaction was separated into a water layer and a butanol layer by centrifugation, and 800 μl of the supernatant (butanol layer) containing the reaction product [ 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. 600 μl of the supernatant was taken and a liquid scintillation counter (LSC) was used to determine the amount of radioactivity (Disintegrations per minute (DPM)). 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 ㎍ / ㎖, the final concentration of the compounds of Formula 1, Formula 2 and Formula 3 prepared in Example 2, HGPAT1 enzyme activity was measured by treatment at concentrations of 30, 10, 3, 1, 0.3 μg / ml. The inhibition rate of hGPAT1 enzyme activity by the sample was calculated by the following equation.

 T: DPM value of the test sphere in which the sample was placed in the enzyme reaction solution,

 C: DPM value of the control 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, Figure 1a and 1b, water lily root extract, fractions thereof or acyclic polyphenol-based compounds isolated therefrom has excellent hGPAT1 inhibitory activity, and concentration-dependent hGPAT1 inhibitory activity Indicated. In addition, IC50 values, which are the concentrations showing 50% inhibitory activity against hGPAT1 of the compounds of Formulas 1, 2 and 3, were 16.9 μg / ml, 70.3 μg / ml and 59.8 μg / ml, respectively.

Final concentration (㎍ / ㎖) hGPAT1 inhibition rate (%) Methanol extract 30 77.1 100 91.4 Hexane fraction 30 70.9 100 85.8 Chloroform fraction 30 78.0 100 91.9 Butanol fraction 30 82.0 100 94.4 Water fraction 30 75.6 100 84.2

Final concentration (㎍ / ㎖) hGPAT1 inhibition rate (%) IC ₅₀ (μg / ml)
Formula 1 300 99.6 16.9 100 98.60 30 74.9 10 36.8 3 17.1 One 15.3
Formula 2 300 98.8 70.3 100 81.5 30 32.2 10 12.2 3 10.9 One 9.3 (3) 300 96.4 59.8 100 74.5 30 31.8 10 9.4 3 8.6 One 7.5

< Example  6> HepG2  Measurement of triglyceride biosynthesis inhibitory effect in cells

In order to examine 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. HepG2 cells were cultured at ATCC and had minimal essential medium (MEM; 2 mM L-glutamine; and 1.5 g / L sodium bicarbonate, 0.1 mM nonessential amino acids and 1 mM). 10% fetal bovine serum (FBS) and 1% antibiotics (100 U / ml penicillin and 100 g / ml streptomycin) were added to earle`s BSS medium formulated to contain sodium pyruvate. The cells were incubated at 37 ° C. in a 5% CO ₂ incubator. Within the cell hGPAT1 enzyme activity was measured by the amount of triglycerides produced in cultured HepG2 cells, and the effects of enzyme inhibitors according to the decrease rate of triglyceride production after addition of compounds of formula 1, formula 2, and formula 3 as enzyme inhibitors Was determined. Dispense at 1 × 10 ⁶ cells / ml per well in a 24-well dish and incubate for 24 hours, exchange with DMEM (Dulbecco's modified Eagle's medium) without FBS 0.2 mCi of [ ¹⁴ C] acetate ([ ¹⁴ C] acetate) (Amersham) was added and reacted for 6 hours. [ ¹⁴ C] glycerol ([ ¹⁴ C] glycerol) used as another substrate was reacted for 18 hours. The sample was dissolved in dimethyl sulfoxide (DMSO), and the control group in the triglyceride formation reaction was reacted with only dimethyl sulfoxide without adding the sample, and the triglyceride produced was 100. .

To determine the amount of triglycerides produced in the finished cells, [ ¹⁴ C] acetate or [ ¹⁴ C] glycerol, which is not absorbed by the cells and remains in the medium, was removed and phosphate-buffer-saline, After removing once with PBS), 0.5 ml of an extraction solvent (hexane: isopropanol = 3: 2, v: v) 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 after removing the extraction solvent, total fat was dissolved in an organic solvent (chloroform: methanol = 2: 1) and thin layer chromatography (TLC: silica gel 60F ₂₅₄ , thickness: 0.5 mm, Merck) was developed on a developing solvent (hexane: 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 ml of 0.1 N sodium hydroxide and used to measure the protein concentration of the cells used for the reaction. The amount of radioactivity of triglycerides was measured, and the experimental values between the test groups were corrected based on the value obtained by dividing the protein concentration into experimental values, thereby calculating the triglyceride production rate by the water extract and fraction 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 ㎍ / ml 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 hexane fraction was 16.7%, chloroform fraction 27.3%, butanol fraction 27.4%, water fraction 35.6% inhibition.

Compound (μg / ml) Substrate used Triglyceride production rate (% of control) Control (DMSO) 30 μg / ml 50 [mu] g / ml Methanol extract [ ¹⁴ C] glycerol 100 ± 3.75 82.3 ± 5.1 84.1 ± 3.2 [ ¹⁴ C] acetate 100 ± 0.8 89.14 ± 9.6 98.6 ± 12.8 Hexane fraction [ ¹⁴ C] glycerol 100 ± 3.75 113.2 ± 6.1 83.3 ± 1.2 [ ¹⁴ C] acetate 100 ± 0.8 126.04 ± 0.5 109.2 ± 10.6 Chloroform fraction [ ¹⁴ C] glycerol 100 ± 3.75 99.7 ± 2.0 72.7 ± 11.4 [ ¹⁴ C] acetate 100 ± 0.8 80.19 ± 3.3 91.23 ± 13.5 Butanol fraction [ ¹⁴ C] glycerol 100 ± 3.75 72.5 ± 6.7 72.6 ± 3.3 [ ¹⁴ C] acetate 100 ± 0.8 71.93 ± 5.2 66.12 ± 4.6 Water fraction [ ¹⁴ C] glycerol 100 ± 3.75 67.5 ± 0.4 64.4 ± 2.7 [ ¹⁴ C] acetate 100 ± 0.8 82.3 ± 1.4 75.65 ± 5.9

< Example  7> water lily root extract or Butanol Fraction L6  cell( rat skeletal  myoblast cell line Measuring the effect of increasing glucose intake

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% propagated throughout the medium followed by 0.2% FBS DMEM (4 mM L-glutamine, 1.5 g / L sodium bicarbonate, 1 g / L glucose) medium was incubated for 24 hours. Under this condition, cells in myoblastes will differentiate into myotubes. Treatment of differentiated L6 cells with insulin and other water extract roots and fractions 30 ㎍ / ㎖ and Formula 1, Formula 2, Formula 3 and Formula 4 at a concentration of 30 uM, respectively (for 1 hour, 3 hours, 6 hours) After incubation for 30 minutes in Krebs-Ringer Buffer (KRB). Cells incubated in KRB were labeled with 0.1 mM 2-deoxy-D-glucose and labeled 2-deoxy-D- [14C] glucose, 0.2 μCi / ml) and reacted for 10 minutes. KRB buffer was removed from the finished cells, 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 water lily root extract and butanol fractions were treated for 6 hours at a concentration of 30 ㎍ / ㎖, the glucose intake was significantly increased compared to the control (DMSO). 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 (DMSO).

< 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 into 7 weeks old male ICR mice (Korean Biolink, Chungbuk Negative) and assigned to 5 animals per group at 8 weeks of age after 1 week of acclimatization. Used for. In the control group, only 0.5% carboxymethyl cellulose (CMC) solution was administered, and the test group was orally administered with the dose of 1,000 mg / kg and 500 mg / kg, respectively, of the water lily root extract of the present invention prepared in Example 1 above. 2 weeks after the toxicity was observed.

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 organs in the thoracic and abdominal cavity that were observed at necropsy. Therefore, it was found that the water lily root extract did not show toxicity due to single oral administration of 1,000 mg / kg and 500 mg / kg, respectively.

< Manufacturing example  1> Sanje  Produce

0.1 g of the water lily root extract of <Example 1>

Lactose 1.5 g

Talc 0.5 g

The above ingredients were mixed and filled in an airtight container to prepare powders.

< Manufacturing example  2> Preparation of tablets

0.1 g of the water lily root fraction of <Example 1>

Lactose 7.9 g

1.5 g of crystalline cellulose

0.5 g of magnesium stearate

After mixing the above ingredients, tablets were prepared by direct tableting method.

< Manufacturing example  3> Preparation of capsules

&Lt; Example 2 > 0.1 g of the compound of Formula 1

5 g of corn starch

4.9 g of carboxy cellulose

After mixing the above components to prepare a powder, the powder was filled in a hard capsule according to a conventional preparation method of a capsule to prepare a capsule.

< Manufacturing example  4> Preparation of injection

0.1 g of a compound of Formula 2 of <Example 2>

Appropriate sterile distilled water for injection

pH adjuster

(2 ml) per ampoule according to the usual injection preparation method.

< Manufacturing example  5> Liquid  Produce

0.1 g of a compound of Formula 3 of <Example 2>

10 g of isomerized sugar

5 g of mannitol

Purified water

Each component was dissolved in purified water in accordance with a conventional method for producing a liquid agent, and the lemon flavor was added in an appropriate amount, followed by mixing the above components. Then, purified water was added thereto to adjust the total volume to 100 ml, and then filled in a brown bottle to sterilize to prepare a liquid.

< Manufacturing example  6> Manufacture of health food

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

Vitamin mixture proper amount

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

0.13 mg of vitamin

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

0.2 μg of vitamin B12

Vitamin C 10 mg

10 μg biotin

Nicotinic Acid 1.7 mg

Folic acid 50 mg

Calcium Pantothenate 0.5mg

Mineral mixture

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dibasic calcium phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

< Manufacturing example  6> Manufacture of health drinks

1000 mg of water lily root extract of <Example 1>

Citric acid 1000 mg

100 g oligosaccharides

Plum concentrate 2 g

1 g of taurine

Add 900 ml of purified water

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was stirred and heated at 85 for about 1 hour. The resulting solution was filtered and sterilized in a sterilized 2 liter container, And used for manufacturing.

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

Claims (15)

Water lily root ( Nymphaea) Tetragona ) Pharmaceutical composition for the prevention and treatment of metabolic diseases containing the extract as an active ingredient.
The pharmaceutical composition for preventing and treating metabolic diseases according to claim 1, wherein the extract is extracted with water, C ₁ to C ₄ alcohols or a mixture thereof.
The pharmaceutical composition for preventing and treating metabolic diseases according to claim 2, wherein the alcohol is ethanol or methanol.
A pharmaceutical composition for preventing and treating metabolic diseases, comprising a fraction obtained by distilling a water lily root extract using an organic solvent.
The pharmaceutical composition for preventing and treating metabolic diseases according to claim 4, wherein the organic solvent is selected from the group consisting of hexane, chloroform and butanol.
The method of claim 4, wherein the fraction is a hexane fraction, chloroform fraction, butanol fraction or water fraction obtained by systematically fractionating the water lily root extract in the order of hexane, chloroform, butanol and water. Composition.
A pharmaceutical composition for preventing and treating metabolic diseases comprising the polyphenol-based compound of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:
&Lt; Formula 1 >

.
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:
(2)

.
A pharmaceutical composition for preventing and treating metabolic diseases comprising the polyphenol-based compound of Formula 3 or a pharmaceutically acceptable salt thereof as an active ingredient:
(3)

.
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:
&Lt; Formula 4 >

.
The pharmaceutical composition according to any one of claims 7 to 10, wherein the compound of Chemical Formula 1, Chemical Formula 2, Chemical Formula 3 or Chemical Formula 4 is isolated from a water lily root extract.
The group of claim 1, wherein the metabolic disease consists of obesity, type 2 diabetes, dyslipidemia, insulin resistance, hepatic steatosis and nonalcoholic fatty liver. Pharmaceutical composition for the prevention and treatment of metabolic diseases, characterized in that selected from.
Water lily root extract, fractions thereof or polyphenol-based compounds isolated therefrom as an active ingredient metabolic disease prevention and improvement composition for health food.
The method of claim 13, wherein the polyphenol-based compound is a health food composition for preventing and improving metabolic diseases, characterized in that selected from the group consisting of the following formula (1), (2), (3) and (4):
&Lt; Formula 1 >

;
(2)

;
(3)

; And
&Lt; Formula 4 >

.
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 resistance, hepatic steatosis and nonalcoholic fatty liver. Health food composition for preventing and improving metabolic disease.

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