KR101462463B1 - Pharmaceutical composition containing Nymphaea tetragona extract, fractions thereof or isolated polyphenolic compounds for prevention or treatment of metabolic disease - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention and treatment of metabolic diseases of water-borne root extract, fractions thereof or polyphenol compounds separated therefrom, wherein the extract of the water-borne root of the present invention and its fractions are glycerol- 3-phosphate (GPAT), inhibits biosynthesis of triglyceride in cells, and increases insulin-dependent glucose uptake in skeletal muscle cells. Therefore, it is possible to inhibit glycerol-3-phosphate The fractions or polyphenol compounds isolated therefrom are useful for the prevention and treatment of metabolic diseases such as obesity, type 2 diabetes, dyslipidemia, insulin resistance, hepatic steatosis and non-alcoholic fatty liver, . ≪ / RTI >

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for preventing and treating metabolic diseases, which comprises a water-extracting root extract, a fraction thereof, or a polyphenolic compound isolated therefrom as an active ingredient. The present invention relates to a pharmaceutical composition containing Nymphaea tetragona extract, fractions thereof or isolated polyphenolic compounds for prevention or treatment of metabolic diseases disease}

The present invention relates to a composition for preventing or treating obesity, type 2 diabetes, dyslipidemia, insulin resistance, hepatic steatosis, and nonalcoholic fatty liver disease, which comprises as an active ingredient a water extract root, a fraction thereof or a polyphenol compound separated therefrom, and a fatty liver. The present invention also relates to a pharmaceutical composition for preventing and treating metabolic diseases.

Biosynthesis of triglyceride in a multistage reaction is the first step glycerol-3-phosphate acyltransferase is catalyzed by (glycerol-3-phosphate acyltransferase, GPAT), glycerol-3-phosphate in sn (glycerol-3-phosphate) - 1 catalyzes the ester reaction to produce lysophosphatidic acid (LPA) by transferring a fatty acid (fatty acyl-CoA) in the form of acyl-coenzyme A to glycerol-3 Limiting step that regulates the rate of the glycerol-3-phosphate pathway (Bell et al, Annu Rev Biochem 49: 459-487, 1980). The last step is the biosynthesis of triglycerides by diacylglycerol acyltransferase (hereinafter "DGAT").

To date, GPAT has been found to have four isoenzymes in mammalian tissues and is distinguished by their susceptibility to and presence in N-ethylmaleimide (NEM). GPAT3 and GPAT4 present in the endoplasmic reticulum (ER) are highly susceptible to sulfhydryl reagent containing NEM, and mtGPAT1, which is present in the mitochondrial outer membrane (MOM) mtGPAT2 exhibits resistance to NEM and is characterized by the preference of saturated fatty acyl-CoA as a substrate (Gonzanlez-Baro et al., Biochim. Biophys. acta, 1771: 830-838, 2007).

Although mtGPAT accounts for about 10% of the total GPAT activity in most tissues of the human body, it is known that the liver tissue accounts for up to 50% of total GPAT activity and is also active in adipose tissue (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 treatment.

 Among the four isoforms of GPAT, only mtGPAT is known to be affected by diet and exercise. Studies have shown that mtGPAT mRNA expression is increased and mtGPAT activity is increased when high calorie diets are consumed in excess of caloric intake. Similarly, after constant exercise, In mice, the activity of mtGPAT was steadily increased as compared with those of mice that did not exercise at all, resulting in a significant increase in the synthesis of triglyceride in the blood (KuMp et al., 2006).

In rats, selective overexpression of mtGPAT in the liver increased lipid biosynthesis in the liver compared to the control group, but significantly decreased fatty acid oxidation and increased fat and dyslipidemia and insulin resistance, In particular, insulin resistance in the liver has increased significantly (Linden et al., FASEB J , 20: 434-443, 2006; Linden et al., J. Lipid Res. 45: 1279-1288, 2004). This implies that lipid metabolism in the liver is mainly involved in the development of insulin resistance. In particular, mtGPAT can be an attractive molecular target for the development of dyslipidemia and insulin resistance treatments associated with non-alcoholic fatty liver disease, since the incidence of fatty liver is significantly higher in obese patients (Linden et al . , FASEB J , 20: 434-443, 2006).

In a related study, when a high fat, high sugar diet was fed to mtGPAT ^- deficient (mtGPAT ^{- / -} ) mice to induce obesity, CPT-1 and β-oxidation (MtGPAT ^{- / -} ) mice showed a 37% decrease in triglyceride content and a 15% decrease in serum compared to the control group. Very low density lipoprotein (VLDL) Was reduced by 30% and consequently, mtGPAT - / - in the mouse was observed to increase the 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).

Thus, GPAT inhibition may be a point of inhibiting intracellular accumulation of triglycerides. Development of low molecular weight inhibitors that regulate the lipid accumulation and energy metabolism of GPAT is associated with obesity, type 2 diabetes, dyslipidemia, insulin resistance, It may be a strategy to develop a therapeutic agent for metabolic diseases such as hepatic insufficiency and nonalcoholic 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 the IC ₅₀ value was in the range of 25 μM to 350 μM, showing a slight inhibitory activity as an enzyme inhibitor. The enzyme source used here is interpreted as the result of the rat mitochondrial fraction still at an early stage. Patents include Fasgen and Princeton University in the United States, and patent 1, which studies that genetic inhibition of mtGPAT and lipid metabolism enzymes is effective in inhibiting and treating various viral infections (WO 2011/019498).

Meanwhile, the water lily ( Nymphaea tetragona is a plant belonging to the water lily family. It is distributed in Korea, Japan, China, India and eastern Siberia. It is an aquatic plant of many years and many petioles grow in thick and short subterranean stem and bloom on the water. The water lily blossom is used to relieve the heat from hangover, relieve hangover, and cure the acute chronic winds of children. In the private sector, flowers are used as medicines such as hemostasis, tonic and insomnia.

As a waterborne root component, polyphenolic geraniin and 1,2,3,4,6-penta-O-gallo-beta-D-glucose (1,2,3,4,6-penta-O -galloyl-beta-D-glucose, PGG) and is known to inhibit apoptosis induced by radiation (Kang et al., Cell Biol., 2011, 27: 83-94, Biol. Pharm Bull., 2010, 33: 1122-1127).

The inventors of the present invention found that the water extract root extract, fractions thereof and polyphenol compounds separated therefrom effectively inhibited the enzyme activity of mtGPAT1 and inhibited the biosynthesis of triglyceride in the process of searching for an active substance inhibiting mtGPAT1 from natural products , Confirming that it has an effect of preventing and treating metabolic diseases such as obesity, type 2 diabetes, dyslipidemia, insulin resistance, hepaticopathy and non-alcoholic fatty liver by increasing glucose intake in skeletal muscle cells, Thereby completing the invention.

It is an object of the present invention to provide a method for the treatment of nymphaea tetragona extract as an active ingredient for the prevention and treatment of metabolic diseases.

Another object of the present invention is to provide a pharmaceutical composition for the prevention and treatment of metabolic diseases, which comprises a fraction obtained by fractionating a water-extracting root extract with an organic solvent.

Another object of the present invention is to provide a pharmaceutical composition for the prevention and treatment of metabolic diseases, comprising a polyphenol compound represented by the following general formula (1), (2), (3) or (4) or a pharmaceutically acceptable salt thereof as an active ingredient .

≪ Formula 1 >

;

;

(2)

;

;

(3)

; 및

; And

≪ Formula 4 >

Another object of the present invention is to provide a composition for a health food for prevention and improvement of metabolic diseases, which comprises a water extract root, a fraction thereof or a polyphenol compound separated therefrom as an active ingredient.

In order to achieve the foregoing object, the present invention is water lily root (Nymphaea The present invention provides a pharmaceutical composition for preventing and treating metabolic diseases, which contains, as an active ingredient,

The present invention also provides a pharmaceutical composition for preventing and treating metabolic diseases, which comprises a fraction obtained by fractionating a water-extracting root extract with an organic solvent.

The present invention also provides a pharmaceutical composition for preventing and treating metabolic diseases, which comprises the polyphenol compound of the above Chemical Formulas 1, 2, 3 or 4, or a pharmaceutically acceptable salt thereof as an active ingredient .

The present invention also provides a composition for a health food for prevention and improvement of metabolic diseases, which comprises a water extract root extract as an active ingredient.

The present invention also provides a composition for a health food for preventing and improving metabolic diseases, which comprises a fraction obtained by fractionating a water-extracting root extract with an organic solvent.

The present invention also provides a composition for health food for preventing and improving metabolic diseases, which comprises the polyphenol compound represented by the above Chemical Formulas 1, 2, 3 or 4, or a pharmaceutically acceptable salt thereof as an active ingredient do.

The polyphenol compounds isolated from the water extract root extract, fractions thereof or fractions thereof according to the present invention effectively inhibit the activity of mtGPAT1 causing metabolic diseases and inhibit biosynthesis of intracellular triglycerides and insulin-dependent glucose uptake in skeletal muscle cells And thus the polyphenolic compounds of the present invention as well as the water extract root extract or fractions thereof containing the polyphenol compounds of the present invention can be used for the prevention and treatment of obesity, type 2 diabetes, dyslipidemia, insulin resistance, hepaticopathy and non- Can be used for the prevention and treatment of metabolic diseases.

FIG. 1 is a graph showing hGPAT1 inhibitory activity of a compound isolated from water extract methanol extract, butanol fraction and fraction.
FIG. 1A is a graph showing inhibitory activity of hGPAT1 on water extract methanol extract and solvent fraction.
FIG. 1B is a graph showing the hGPAT1 inhibitory activity of the formula (1), (2), (3) or (4).
Fig. 2 is a graph showing the inhibitory effect of methanol extracts of the water-extracting roots and the solvent fraction on neutral fat synthesis in HepG2 cells (human-derived hepatocytes). Fig.
FIG. 3 is a graph showing glucose uptake activity of water extract methanol extract, fractions and isolated compounds.
FIG. 3A is a graph showing glucose uptake activity in L6 cells, which are rat skeletal muscle cells, of methanol extracts and fractions of water-extract roots.
FIG. 3B is a graph showing glucose uptake 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 invention water lily root (Nymphaea The present invention provides a pharmaceutical composition for preventing and treating metabolic diseases, which contains, as an active ingredient,

As used herein, the term " Nymphaea tetragona "refers to all organs of a natural, hybrid or variant water lily, including, for example, roots, branches, stems, leaves and flowers,

The extract may be extracted with a solvent such as water, a C ₁ to C ₄ alcohol, or a mixture thereof. Specifically, the alcohol may be ethanol or methanol, but is not limited thereto.

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

1) extracting the extract of the water lily by adding an extraction solvent;

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

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

In the above method, the cultivation roots of step 1) may be cultivated or commercially available. The roots of the water lily may be those using the water lily roots, but the present invention is not limited thereto.

 As the method for extracting the water-extracting root extract, conventional methods in the art can be used such as filtration, hot water extraction, immersion extraction, reflux cooling extraction and ultrasonic extraction. The extract may be extracted one to five times by a hot water extraction method, More specifically, it may be repeated three times, but is not limited thereto. The extraction solvent may be added to the dried water lily roots 0.1 to 10 times, preferably 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 this method, the vacuum concentration of step 3) may be by using 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 diseases may be selected from the group consisting of obesity, type 2 diabetes, dyslipidemia, insulin resistance, hepatic steatosis and fatty liver. However, the present invention is not limited thereto.

The present invention also provides a pharmaceutical composition for preventing and treating metabolic diseases, which comprises a fraction obtained by fractionating a water-extracting root extract with 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 a hexane fraction, a chloroform fraction, a butanol fraction or a water fraction obtained by systematically fractionating the water extract root extract in the order of hexane, chloroform, butanol and water, but is not limited thereto. The hexane is preferably n-hexane.

The metabolic diseases may be selected from the group consisting of obesity, type 2 diabetes, dyslipidemia, insulin resistance, hepatic steatosis and fatty liver. However, the present invention is not limited thereto.

The present invention also provides a pharmaceutical composition for preventing and treating metabolic diseases, which comprises a polyphenol compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

≪ Formula 1 >

The present invention also provides a pharmaceutical composition for preventing and treating metabolic diseases, which comprises a polyphenol compound represented by the following formula (2) or a pharmaceutically acceptable salt thereof as an active ingredient.

(2)

The present invention also provides a pharmaceutical composition for preventing and treating metabolic diseases, which comprises a polyphenol compound represented by the following formula (3) or a pharmaceutically acceptable salt thereof as an active ingredient.

(3)

The present invention also provides a pharmaceutical composition for preventing and treating metabolic diseases, which comprises a polyphenol compound represented by the following formula (4) or a pharmaceutically acceptable salt thereof as an active ingredient.

≪ Formula 4 >

The compounds of formulas (1), (2), (3) and (4) may be separated from the extract of root extract, but not limited thereto.

The metabolic diseases may be selected from the group consisting of obesity, type 2 diabetes, dyslipidemia, insulin resistance, hepatic steatosis and fatty liver. However, the present invention is not limited thereto.

In order to prepare the water extract root fractions and fractions, the present inventors extracted water roots by pulverizing and immersing them in methanol. The extract was filtered and concentrated under reduced pressure to obtain a methanol extract. To separate the active substances, the water-roots methanol extracts were subjected to solvent fractionation with hexane, chloroform, butanol and water to obtain respective fractions. The human glycerol- - 3-phosphate acyltransferase (hGPAT1) inhibitory activity, the inhibitory activity was better than that of the butanol fraction.

To separate the compounds from the water extract root extract, the present inventors separated the butanol fraction into 8 fractions by reverse phase column chromatography, sequentially increasing the polarity with a methanol / water mixed solvent (see Fractions 1 to 8). Fractions with the highest inhibitory activity were collected and eluted with methanol using Sephadex LH-20 (Sephadex LH20) to separate the active fractions. High-performance liquid chromatography (YMC J'sphere ODS H-80 column, 250 x 20 mm) was performed with collecting the fractions having strong inhibitory activity and flowing a methanol / water mixed solvent at 5 ml / min as an eluting solvent. Four kinds of pure compounds (Formula 1, Formula 2, Formula 3 and Formula 4) were obtained.

The inventors of the present invention measured the structure, molecular weight, molecular formula, mass spectrometry, ¹ H-NMR spectrum and ¹³ C-NMR spectrum of the above compounds in order to clarify the structure of the above four compounds. 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 ] were consistent with the data, non-geranic (geranin), 1,2,3,4,6- penta-O-get Roy-beta -D- glucose (1, 2, D-glucose), 1,2,3,6-tetra- O- gal-beta-D-glucose (1,2,3,6-tetra- O-galloyl-beta-D-glucose, and methyl gallate. The physicochemical properties and chemical structure of the compounds isolated from the water lily roots are as follows.

≪ Formula 1 >

Geranin

(2)

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

(3)

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

≪ Formula 4 >

Methyl gallate

The present inventors cloned the human hGPAT1 cDNA (NCBI accesstion No. NM_020918) into the pFastBac1 vector in order to isolate the mass expression of hGPAT1 (human GPAT1) derived from human body and the hGPAT1 enzyme source, transformed the obtained recombinant bacmid DNA into Sf9 cells Baculovirus containing human hGPAT1 cDNA was assembled and amplified. Cells were harvested and homogenized using an ultrasonic cell scraper. Only the supernatant was taken and centrifuged. The obtained precipitate was suspended in sucrose buffer and used as an enzyme source of mitochondrial hGPAT1 (mitochondrial hGPAT1). The supernatant obtained by centrifuging the supernatant after removing the precipitate was suspended in a sucrose buffer and used as a microsomal hGPAT1 enzyme source.

In order to measure the inhibitory effect of human hGPAT1 on the activity of the microsomal hGPAT1 enzyme, its activity is lost by N-ethylmaleimide (NEM). When measuring only mitochondrial hGPAT1 activity, Ethylmaleimide was pretreated to disrupt microsomal hGPAT1 activity and then the experiment proceeded. HGPAT1 mitochondrial activity of the enzyme in the resulting reaction product ^[14 C] resources phosphatides diksan ^([14 C] Lysophosphatidic acid) was measured by the amount of radiation. The activity of mitochondrial hGPAT1 was measured by pretreatment of 2 mM N-ethylmaleimide in an ice bath for 15 minutes before the experiment to inactivate the activity of microsomal hGPAT1, and the activity of microsomal hGPAT1 The activity of the microsomal hGPAT1 enzyme source was corrected by measuring the ratio of total hGPAT1 activity and microsomal hGPAT1 enzyme activity by pre-treating the microsomal hGPAT1 enzyme source with N-ethylmaleimide (2 mM).

The substrates used for the enzyme reaction were [ ¹⁴ C] glycerol-3-phosphate (1.8 μM) and palmitoyl-CoA (100 μM) The reaction was stopped by water. The reaction mixture was separated into a water layer and a butanol layer by centrifugation and an upper layer (butanol layer) containing [ ¹⁴ C] lysophosphatidic acid as a reaction product was taken. The supernatant was separated into water layer and butanol layer by centrifugation with the same volume of water, and 600 μl of the supernatant was removed and the amount of radioactivity (Disintegrations per minute, DPM) was measured with a liquid scintillation counter (LSC) Respectively. The extracts, fractions and the compounds of Formulas (1), (2) and (3) prepared in Examples 1 and 2 were each treated to measure the activity of hGPAT1 enzyme. As a result, the water-extracting root extract, its fractions or the acyclic polyphenol compounds isolated therefrom were excellent in hGPAT1 inhibitory activity and hGPAT1 inhibitory activity in a concentration-dependent manner (see Table 1, Table 2, FIG. 1A and FIG. 1B ).

In order to examine the effects of the compounds of the present invention on the triglyceride synthesis in cells, the inhibitory activity of the triglyceride biosynthesis in the cells on the two substrates was measured using human hepatocyte HepG2 cells. As a result, as shown in Table 3, when the water extract and fraction were treated at a concentration of 50 μg / ml and [ ¹⁴ C] glycerol was used as a substrate, each fraction inhibited the production of triglyceride in the cells (see Table 3) ).

To measure glucose uptake in L6 cells (rat skeletal myoblast cell line), differentiated L6 cells were treated with insulin, a water extract root or fraction, and the compounds of Formulas 1, 2, 3 and 4, (Krebs-Ringer Buffer, KRB). 2-deoxy-D-glucose and labeled 2-deoxy-D- [14C] glucose, 0.2 μCi / ml ) And allowed to react for 10 minutes. After the completion of the reaction, the glucose level was measured using a scintillation counter. As a result, treatment with water extract of roots or butanol fraction significantly increased glucose uptake compared to control group (DMSO). In addition, treatment of Formulas 1, 2, 3 and 4 significantly increased glucose uptake compared to control (DMSO).

In order to measure glucose uptake in L6 cells (rat skeletal myoblast cell line) of the water extract root or butanol fraction, differentiated L6 cells were treated with insulin, a water extract root extract and a butanol fraction, and then Krebs-Ringer buffer -Ringer Buffer, KRB). 2-deoxy-D-glucose and labeled 2-deoxy-D- [ ¹⁴ C] glucose, 0.2 μCi / ml) and allowed to react for 10 minutes. After the completion of the reaction, the glucose level was measured using a scintillation counter. As a result, treatment with water extract of roots or butanol fraction significantly increased glucose uptake and insulin dependence compared with control (DMSO).

In order to examine the oral acute toxicity of the extract of root of the present invention, the following experiment was conducted using a mouse. Seven weeks old male ICR mice were divided into a control group and a test group. The control group was administered with only 0.5% carboxymethyl cellulose (CMC) solution, Were orally administered at a dose of 1,000 mg / kg and 500 mg / kg, respectively, of the extract of the present invention prepared in Example 1, and then observed for toxicity for 2 weeks. As a result, none of the animals died in all groups, and no animals showed any unusual behavior or toxic symptoms. In addition, weight gain was increased in all groups, and no abnormal lesions or abnormalities were found in all the organs in thoracic cavity and abdominal cavity observed at autopsy.

Therefore, the water extract of roots of the present invention, its fractions, and the polyphenol compounds isolated from the fractions effectively inhibit the activity of mtGPAT1 causing metabolic diseases, and inhibit biosynthesis of intracellular triglycerides and insulin-dependent The extracts of roots or their fractions of the present invention including the polyphenol compounds according to the present invention can be used for the treatment of obesity, type 2 diabetes, dyslipidemia, insulin resistance, hepaticopathy and non-alcoholic fatty liver Can be used as an active ingredient of a pharmaceutical composition for the prevention and treatment of metabolic diseases.

The composition of the present invention may contain 0.1 to 99.9% by weight of the extract of the seedlings of the present invention or a fraction thereof as an active ingredient, and may include a pharmaceutically acceptable carrier, excipient or diluent.

The compositions of the present invention may be of various oral or parenteral formulations. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like, which may contain one or more excipients such as starch, calcium carbonate, sucrose or lactose lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The composition of the present invention may be administered orally or parenterally, and it is preferable to select the intraperitoneal, rectal, rectal, intravenous, intramuscular, subcutaneous, intrauterine or intracerebral injection methods for parenteral administration, It is used for external skin.

The dosage of the composition of the present invention varies depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate and severity of disease, and the daily dose is the amount of water- Kg, preferably 30 to 500 mg / kg, more preferably 50 to 300 mg / kg, and may be administered 1 to 6 times a 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 composition according to the present invention in a pharmaceutically effective amount.

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

The metabolic diseases may be selected from the group consisting of obesity, type 2 diabetes, dyslipidemia, insulin resistance, hepaticopathy, and non-alcoholic fatty liver, but the present invention is not limited thereto.

The pharmaceutically effective amount is 0.0001 to 100 mg / kg, and 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 at the time of clinical administration and can be administered orally or parenterally in the case of parenteral administration by intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine injection, intracerebral injection, And may be used in the form of a general pharmaceutical preparation.

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

In the present invention, "administering" means introducing a predetermined substance into a patient in any appropriate manner, and the administration route of the substance can be administered through any conventional route so long as it can reach the target tissue. But are not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrathecal, rectal. In addition, the pharmaceutical composition may be administered by any device capable of moving the active substance into the target cell.

In the present invention, "administering" means introducing the composition of the present invention into a pancreatic cancer cell by "systemic delivery" or "topical delivery "."Systemicdelivery" refers to delivery which results in a broad-spectrum biodistribution of compounds within an organism. Some administration techniques may lead to systemic delivery of certain compounds, while others may not. Systemic delivery means that a useful, preferably therapeutically effective amount of a compound is exposed to the majority of the body. Generally, in order to obtain a global bio-distribution, it is desirable that the compound not be rapidly degraded or removed prior to reaching the diseased site far from the site of administration (e.g., by first passage organ (liver, lung, etc.) In the blood) is required. As used herein, "topical delivery" refers to direct delivery of a compound to a target site in an organism. For example, the compound may be delivered locally by direct injection into a diseased site, such as a tumor or other target site, e.g., a target organ such as the pancreas.

The present invention also provides a composition for a health food for prevention and improvement of metabolic diseases, which comprises a water extract root extract as an active ingredient.

The present invention also provides a composition for a health food for preventing and improving metabolic diseases, which comprises a fraction obtained by fractionating a water-extracting root extract with an organic solvent.

In addition, the present invention provides a composition for health food for preventing and improving metabolic diseases, which comprises a polyphenolic compound or a pharmaceutically acceptable salt thereof as an active ingredient.

The polyphenol compound may be selected from the group consisting of the compounds represented by Chemical Formulas 1, 2, 3 and 4, but is not limited thereto.

The metabolic diseases may be selected from the group consisting of obesity, type 2 diabetes, dyslipidemia, insulin resistance, hepatic steatosis and fatty liver. However, the present invention is not limited thereto.

In the present invention, it has been confirmed that the water extract root extract, its fractions and the polyphenol compounds isolated from the fractions effectively inhibit the activity of mtGPAT1 which causes metabolic diseases. Therefore, the polyphenol compounds of the present invention, The fraction thereof can be used as an active ingredient of a composition for a health food for preventing and ameliorating metabolic diseases such as obesity, type 2 diabetes, dyslipidemia, insulin resistance, hepaticopathy and non-alcoholic fatty liver.

The functional food of the present invention may contain various flavors or natural carbohydrates as an additional ingredient. 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 may be selected from the range of 0.01 to 0.04 part by weight, specifically about 0.02 to 0.03 part 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 may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, , A carbonating agent used in carbonated drinks, and the like. In addition, the functional food of the present invention may contain flesh for the production of natural fruit juice, fruit juice drink and vegetable drink. These components may be used independently or in combination. The proportion 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 present invention will be described in more detail with reference to Examples and Preparation Examples.

However, the following examples and preparative examples are merely illustrative of the present invention, and the present invention is not limited to the following examples and preparative examples.

< Example  1> water lily root extract and Fraction  Produce

5 kg of dried water lily roots were pulverized and immersed in 25 L of methanol and extracted twice. The extract was filtered and concentrated under reduced pressure to obtain about 199 g of a methanol extract. In order to isolate the active substance, 199 g of the methanol extract of the water lily root was suspended in 1000 ml of distilled water, and the fractions were separated by solvent fractionation with hexane, chloroform, butanol and water. As a result of measuring the hGPAT1 enzyme inhibition activity of the above fractions, the inhibitory activity was better than that of the butanol fraction.

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

The butanol fraction (about 25 g) was subjected to reversed-phase column chromatography using sequential polarity increasing with 20%, 30%, 40%, and 50% methanol / (Fractions 1 to 8).

Fractions with the highest inhibitory activity were collected and eluted with methanol using Sephadex LH-20 (Sephadex LH20) to separate the active fractions. High-performance liquid chromatography (YMC J'sphere ODS H-80 column, 250 x 20 mm) was carried out by collecting fractions with strong inhibitory activity and eluting with a 90% methanol / water mixed solvent at 5 ml / Finally, four pure compounds (Formula 1, Formula 2, Formula 3 and Formula 4) were obtained.

< Example  3> Identification of compound structure

The compound was analyzed for the properties of a substance, molecular weight, molecular formula, mass spectrometry, ¹ H-NMR spectrum and ¹³ C-NMR spectrum, and the results are shown 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 ] were consistent with the data, non-geranic (geranin) (I), 1,2,3,4,6-penta-O-get Roy-beta -D- glucose ( (1, 2, 3, 4, 6-penta- O- galloyl-beta-D- glucose), 1,2,3,6-tetra-O- gallo-beta-D- 2,3,6-tetra-O-galloyl-beta-D-glucose (Formula 3) and methyl gallate (Formula 4). The physicochemical properties and chemical structure of the compounds isolated from the water lily roots are as follows.

1) Appearance: light brown solid (pale brown solid)

2) Molecular weight: 952

3) Molecular formula: C ₄₁ H ₂₈ O ₂₇

4) ESI-Mass Spec .: m / z = [M + Na] &lt; ⁺ &gt;, 975.06

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

6) ¹³ C-NMR (100 MHz, CD ₃ OD,? (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, , 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, 144.90, 144.96, 145.13, 145.23 , 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: light brown solid (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 (400MHz} , 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 , 6), 6.22 (1H, d, J = 9.5 Hz, glu-H-1).

6) ¹³ C-NMR (100 MHz, CD ₃ OD,? (Ppm)): 167.9 (2-Gall-C-7), 167.3 ), 166.9 (4-Gall-C-7), 166.2 (6-Gall-C-7), 146.5 146.4 (3-Gall-C-3,5), 140.8 (1-Gall-C-4), 146.3 140.4 (2-Gall-C-4), 140.3 (3-Gall-C-4), 140.1 C-1), 120.4 (2-Gall-C-1), 120.3 (3-Gall-C-1), 120.2 C-2,6), 110.4 (2-Gall-C-2,6, 3-Gall- C-2,6), 93.8 (glu-C-1), 74.4 (glu-C-3), 74.1 4), 63.1 (glu-C-6).

1) Appearance: light brown solid (pale brown solid)

2) Molecular weight: 788

3) Molecular formula: C ₃₄ H ₂₈ O ₂₂

4) ESI-Mass Spec .: m / z = [M + Na] ⁺ , 811

^{5) 1 H-NMR (400MHz} , 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 (100MHz} , 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 ), 146.2 (2-Gall-C-3), 121.2 (6-Gall-C-1), 120.8 C-2,6,6-Gall-C-2,6), 110.3 (3-Gall-C-2,6,6- C-2), 68.7 (glu-C-4), 73.6 (glu- 64.4 (glu-C-6).

1) Appearance: white powder

2) Molecular weight: 184

3) Molecular formula: C8H8O5

4) 1 H-NMR (400MHz, CD 3 OD,? (Ppm)): 7.41 (1H, s, H-2), 7.75 1H, s, ester methyl)

5) 13 C-NMR (100 MHz, CD 3 OD,? (Ppm)): 121.5 (C-1), 110.6 (C-2), 149.9 , 118.7 (C-6), 168.2 (C-7), 56.8 (methoxy methyl), 49.5 (ester methyl).

< Example  4> Human origin hGPAT1  ( human GPAT1 ) &Lt; / RTI &gt; Enzyme source  detach

Mitochondrial protein isolated from sf9 cells overexpressing human GPAT1 (hGPAT1) was used as an enzyme source. The hGPAT1 cDNA (NCBI accesstion No. NM_020918) was cloned into the pFastBac1 vector (Invitrogen), and the resulting recombinant bacmid DNA was transformed into Sf9 cells, and baculovirus containing human hGPAT1 cDNA was assembled and amplified. Sf9 cells (1 x 10 6 cells / ml) were infected with 10 MOI of virus, and after 48 hours, the cells were recovered by centrifugation and homogenized buffer (250 mM sucrose, 10 mM Tris ), 1 mM EDTA) and homogenized using an ultrasonic cell crusher. The supernatant was separated by centrifugation (600 g, 15 minutes) and centrifuged at 8,000 g for 15 minutes. The resulting precipitate, the mitochondrial protein fraction, was dissolved in sucrose buffer (250 mM sucrose, 10 mM Tris 1 mM EDTA, 1 mM DTT), and the protein concentration was determined by the Bradford method. The protein concentration was determined at -80 ° C and used in the experiment.

< Example  5> Human origin hGPAT1 Of active inhibition

The activity of the hGPAT1 enzyme was measured by the amount of [14C] lysophosphatidic acid, the reactant produced by the mitochondrial hGPAT1 enzyme source. The activity of mitochondrial hGPAT1 was maintained by treatment of ethylmaleimide (NEM) with hGPAT1 compared to other isozymes. According to the previous study, 2 mM N-ethylmaleimide was added to the hGPAT1 enzyme source Min in an ice bath to inactivate the activity of homozygous enzymes other than hGPAT1.

The substrate used for the enzyme reaction was [ ¹⁴ C] glycerol-3-phosphate (1.8 μM) and palmitoyl-CoA (100 μM) , 4 mM MgCl ₂ , 8 mM NaF, 2 mg / ml BSA) at 26 ° C for 20 minutes, and the reaction was stopped by water-saturated butanol and water. The reaction product was separated into a water layer and a butanol layer by centrifugation and 800 μl of an upper layer (butanol layer) containing [ ¹⁴ C] lysophosphatidic acid as a reaction product was taken. The supernatant was separated into water layer and butanol layer by centrifugation with the same volume of water, and 600 μl of the supernatant was removed and the amount of radioactivity (Disintegrations per minute, DPM) was measured with a liquid scintillation counter (LSC) Respectively. The final concentrations of the extracts and fractions obtained from the water extracts and fractions prepared in Examples 1 and 2 were treated at a concentration of 30,100 μg / ml, and the final concentrations of the compounds of Formulas 1, 2 and 3 prepared in Example 2 were 100, 30, 10, 3, 1, and 0.3 / / ml to measure hGPAT1 enzyme activity. The inhibition rate of hGPAT1 enzyme activity by the sample was calculated by the following equation (1).

 T: DPM value of the test solution containing the sample in the enzyme reaction solution,

 C: DPM value of the control sample not containing the sample in the enzyme reaction solution,

 B: DPM value of the control sample without the enzyme source added.

As a result, as shown in Tables 1, 2, 1A and 1B, the water extract root extract, fractions thereof or acyclic polyphenol compounds separated therefrom showed excellent hGPAT1 inhibitory activity and hGPAT1 inhibitory activity in a concentration- Respectively. The IC50 values of the compounds of the formulas (1), (2) and (3), which exhibit 50% inhibitory activity against hGPAT1, were 16.9 ㎍ / ㎖, 70.3 ㎍ / ㎖ and 59.8 ㎍ / ㎖ respectively.

Final concentration ([mu] g / ml) hGPAT1 Inhibition (%) 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 ([mu] g / ml) hGPAT1 Inhibition (%) IC ₅₀ ([mu] g / ml)
Formula 1 300 99.6 16.9 100 98.60 30 74.9 10 36.8 3 17.1 One 15.3
(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 the inhibitory effect of trastuzumab biosynthesis in cells

In order to examine the effects of the compounds of the present invention on the triglyceride synthesis in cells, the inhibitory activity of the triglyceride biosynthesis in the cells on the two substrates was measured using human hepatocyte HepG2 cells. HepG2 cells were purchased from ATCC and cultured in minimal essential medium (MEM; 2 mM L-glutamine; 1.5 g / L sodium bicarbonate, 0.1 mM nonessential amino acids and 1 mM Briefly, 10% fetal bovine serum (FBS) and 1% antibiotic (100 U / ml penicillin and 100 g / ml streptomycin) were added to the earle`s BSS medium supplemented with sodium pyruvate And cultured in a 5% CO <sub>2</sub> incubator at 37 ° C. Intracellular The activity of hGPAT1 enzyme was measured by the amount of triglyceride produced in cultured HepG2 cells. After the addition of the compounds of formulas (1), (2) and (3) as enzyme inhibitors, the effect of enzyme inhibitor . The cells were cultured in a 24-well culture dish at a cell number of 1 × 10 <sup>6</sup> cells / ml per well and cultured for 24 hours. Then, the medium was replaced with DMEM (Dulbecco's modified Eagle`s medium) [ ^14C ] acetate ([ ^14C ] acetate) (Amersham) was added and reacted for 6 hours. In the case of using a different substrate ^[14 C] glycerol ^([14 C] glycerol), the reaction was carried out for 18 hours. Samples were dissolved in dimethyl sulfoxide (DMSO), and the control in the neutral fat production reaction was carried out with dimethyl sulfoxide alone without adding the sample, and the production rate of the produced triglyceride was 100 .

[ ¹⁴ C] acetate or [ ¹⁴ C] glycerol, which is the substrate remaining in the medium, is not absorbed into the cells and is removed by phosphate-buffer-saline, After removing once with PBS, 0.5 ml of extraction solvent (hexane: isopropanol = 3: 2, v: v) was added to extract whole fat including neutral fat. After extracting with 0.5 ml of extraction solvent twice for 30 minutes, 1 ml of the extract was concentrated through nitrogen gas. After removing the extraction solvent, whole fat was dissolved in an organic solvent (chloroform: methanol = 2: 1) and thin layer chromatography (TLC: (hexane: diethyl ether: acetic acid = 80: 20: 1, v: v: v) on a silica gel 60F ₂₅₄ , thickness: 0.5 mm, Merck). 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] radioactivity was measured. After the extraction, the remaining cells were dissolved in 0.3 ml of 0.1 N sodium hydroxide and used to measure the protein concentration of the cells used in the reaction. The amount of neutral fat produced by the water extracts and fractions of the present invention was calculated by adjusting the experimental error between the test groups by measuring the radioactivity of neutral fat and dividing the protein concentration by experimental values.

As a result, when the water extract and fraction were treated at a concentration of 50 μg / ml and [ ¹⁴ C] glycerol was used as a substrate, the production of triglyceride in the cells was found to be 15.9% for the methanol extract and 15.9% for the hexane fraction 16.7%, chloroform fraction 27.3%, butanol fraction 27.4% and water fraction 35.6%, respectively.

Compound (쨉 g / ml) Substrate used Percentage of triglyceride production (% of control) The control (DMSO) 30 [mu] g / 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 ) Increase in glucose uptake

L6 myoblasts were cultured in DMEM (4 mM L-glutamine, 1.5 g / L sodium hydrogencarbonate, 4.5 g / L glucose) medium containing 10% FBS. Cells cultured on 24-well plates were washed twice with PBS in a state where 80% of the cells were grown on the whole surface of the medium, and then cultured in 0.2% FBS DMEM (4 mM L-glutamine, 1.5 g / L sodium hydrogencarbonate, 1 g / L glucose) medium and cultured for 24 hours. Under these conditions, cells in the myoblastes state differentiate into myotubes. The differentiated L6 cells were treated with 30 μg / ml of insulin and other water extracts and fractions and 30 μM of 1, 2, 3 and 4, respectively, for a predetermined time (1 hour, 3 hours, 6 hours) And then cultured in Krebs-Ringer Buffer (KRB) for 30 minutes. To the cells cultured in KRB, 0.1 mM 2-deoxy-D-glucose and labeled 2-deoxy-D- [14C] glucose, 0.2 μCi / ml) and allowed to react for 10 minutes. KRB buffer was removed from the cells after the reaction, washed three times with KRB buffer, and the cells were lysed with 1 M sodium hydroxide (NaOH). Cells dissolved in 1 M sodium hydroxide were measured with a scintillation counter to determine glucose uptake.

As a result, the treatment with water extract of roots and butanol fraction at 30 ㎍ / ㎖ for 6 hours significantly increased glucose uptake compared to control (DMSO). In addition, treatment with the compounds of formulas (1), (2), (3) and (4) at a concentration of 30 uM for 6 hours significantly increased glucose uptake compared to the control group (DMSO).

< Example  8> Acute Toxicity of Water Extracts

In order to examine the oral acute toxicity of the extract of root of the present invention, the following experiment was conducted using a mouse. Seven weeks old male ICR mice (BioLink, Chungbuk province) were introduced into the experimental animals as specific pathogen free (SPF) animals. After 8 weeks of age, they were assigned to 5 mice per group Lt; / RTI &gt; In the control group, only 0.5% carboxymethyl cellulose (CMC) solution was administered, and in the test group, the extract of root of the present invention prepared in Example 1 was orally administered at a dose of 1,000 mg / kg and 500 mg / kg, respectively And then observed for toxicity for 2 weeks.

As a result, none of the animals died in all groups, and no animals showed any unusual behavior or toxic symptoms. In addition, weight gain was increased in all groups, and no abnormal lesions or abnormalities were found in all the organs in thoracic cavity and abdominal cavity observed at autopsy. Therefore, it was found that the water extract root extract did not show toxicity by single oral administration of 1,000 mg / kg and 500 mg / kg to mice, respectively.

< Manufacturing example  1> Sanje  Produce

&Lt; tb > &lt; tb &gt; &lt; tb &gt;

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 < RTI ID = 0.0 >

Lactose 7.9 g

Crystalline cellulose 1.5 g

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

Corn starch 5 g

4.9 g of carboxycellulose

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

The compound of Formula 2 of Example 2

Sterile sterilized 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 the compound of Formula 3 of Example 2

10 g per isomer

5 g mannitol

Purified water quantity

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 to adjust the total volume to 100 ml, and the solution was filled in a brown bottle and sterilized to prepare a liquid preparation.

< Manufacturing example  6> Manufacture of health food

100 mg of the compound of formula 4 in Example 2

Vitamin mixture quantity

70 [mu] g of vitamin A acetate

Vitamin E 1.0 mg

0.13 mg of vitamin

0.15 mg of vitamin B2

0.5 mg vitamin B6

0.2 [mu] g vitamin B12

10 mg vitamin C

Biotin 10 μg

Nicotinic acid amide 1.7 mg

50 mg of folic acid

Calcium pantothenate 0.5 mg

Mineral mixture quantity

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

15 mg of potassium phosphate monobasic

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

100 mg of calcium carbonate

24.8 mg of magnesium chloride

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 the extract of root extract of Example 1

Citric acid 1000 mg

100 g of oligosaccharide

Plum concentrate 2 g

Taurine 1 g

Purified water was added to a total of 900 ml

The above ingredients were mixed according to the usual health drink manufacturing method, and the mixture was stirred and heated at 85 for about 1 hour. The resulting solution was filtered to obtain a sterilized 2-liter container, which was sealed and sterilized, And used for manufacturing.

Although the composition ratio is relatively mixed with the ingredient suitable for the favorite drink, it is also possible to arbitrarily modify the blending ratio according to the regional or national preference such as the demand class, demand country, use purpose, and the like.

Claims (15)

Pharmaceuticals for the prophylaxis and treatment of diseases selected from the group consisting of obesity, hepatic steatosis and non-alcoholic fatty liver comprising the hexane, chloroform and butanol fractions of the extract of Nymphaea tetragona as active ingredients Composition.
The method of claim 1, wherein the extract is extracted with a solvent that is water, a C ₁ to C ₄ alcohol, or a mixture thereof. A pharmaceutical composition.
3. The pharmaceutical composition according to claim 2, wherein the alcohol is ethanol or methanol. 4. A pharmaceutical composition for preventing and treating diseases selected from the group consisting of obesity, gonadal hyperplasia and non-alcoholic fatty liver.
delete delete The method according to claim 1, wherein the fraction is a hexane fraction, a chloroform fraction or a butanol fraction obtained by fractionating the water extract root extract in the order of hexane, chloroform and butanol. A pharmaceutical composition for the prevention and treatment of selected diseases.
delete delete delete delete delete delete A composition for a health food for the prevention and improvement of diseases selected from the group consisting of obesity, gonadal hyperplasia and non-alcoholic fatty liver which contains hexane, chloroform and butanol fractions of a water-extracting root extract as an active ingredient. delete delete

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