KR20140110552A - Pharmaceutical compositions for the prevention and treatment of obesity containing biological active materials from Curcuma aromatica Salisb. extracts - Google Patents

Abstract

The present invention relates to a pharmaceutical composition comprising a Curcuma longa derived physiologically active material as an active ingredient for preventing and treating obesity. Dimethoxycurcumin and curcumin among diarlyheptanoid based compounds, which are Curcuma longa derived physiologically active material of the present invention, show excellent effects of inhibiting lipase, thus the Curcuma longa derived physiologically active material of the present invention can be usefully applied in preventing and treating obesity.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for prevention and treatment of obesity, which comprises a physiologically active substance derived from ganoderma, as an active ingredient. extracts}

The present invention relates to a pharmaceutical composition for the prevention and treatment of obesity, which comprises an organic solvent fraction isolated from an urokinase extract and a diarylheptanoid-based compound separated therefrom as an active ingredient.

Recently, with the improvement of industrial development and income level, along with the westernization of lifestyle such as eating habits and eating habits, chronic diseases and geriatric patients are increasing rapidly. One of the causes is known as obesity. Obesity is an energy metabolism abnormality caused by an imbalance in metabolism of food and food, resulting in excessive accumulation of triglyceride in adipocytes. The most common cause of obesity is increased body weight or body fat accumulation due to ingestion of food containing high energy or high fat, lack of exercise, or accumulation of fat in the body. Recently, however, due to neuroendocrine abnormalities, drugs, genetic factors and biochemical abnormalities (Bray GA, Popkin BM. (1998) Dietary fat intake affect obesity Am . J. Clin . Nutr . , 68, 1157-1173 Bra, GA Popkin BM 1999 Dietary fat affects obesity rate. Am . J. Clin . Nutr. , 70, 572-573). Obesity has been reported to increase not only in appearance but also in weight gain and in severe adult diseases such as hypertension, type 2 diabetes, heart disease, stroke, arthritis, arteriosclerosis and cancer (Freedman DS, Serdula MK, Rexrode KM, Manson JE, Hennekens CH. (1996) Obesity and cardiovascular disease, Curr. Opin. Cardiol., 11, 490-495).

As a principle of treatment of obesity, diet and exercise are the most appropriate methods, but in recent years development of appetite suppressant, body fever accelerator, and body absorption inhibitor has been developed and the utility of anti-fatigue therapy has been attracting attention. Among them, pancreatic lipase inhibitor ) Are interested. Lipase inhibitors act as key enzymes that degrade triglycerides into 2-monoacylglycerols and fatty acids (Bitou N, Nimomiya M, Tsjita T, Okuda H. 1999) Screening of lipase inhibitors from marine algae. Lipids, 34, 441-445). Representative lipase inhibitors include Streptomyces It is known that tetrahydrolipstatin (or listat), a derivative of lipstatin derived from toxitricini, is most effective in inhibiting about 30% of the fat ingested (Drent ML, Larsson I, William-Olsson T, (1995) Orlistat (RO 18-0647), a lipase inhibitor, in the treatment of human obesity: a multiple dose study, Int J J. Obesity, 19, 221-226; Hadvay P, Lengsfeld H, Wolter H. (1988) Inhibition of pancreatic lipase in vitro by covalent inhibitor tetrahydrolipstatin, Biochem J. 256, 357-361) . Despite this efficacy, orlistat has been known to have side effects such as gastrointestinal disorders, hypersensitivity, biliary secretion, and lipid-soluble vitamin absorption inhibition (Peter C, Williams G. (2001) Drug treatment of obesity: from past failures to future successes Br. J. Clin. Pharmacol., 51, 135-141). Recently, a number of studies have been conducted on the development of pancreatic lipase inhibitors from natural materials without side effects (Yamamoto M, Shimura Y, Iyoh M Egawa, S Ionue. (2000). Anti-obesity effects of lipase inhibitors And a lipid-lowering enzyme from Salvia officinalis, Salacia reticulata, and oolong tea, as well as a high-fat diet such as CT-II, an extract from edible herbs, Nomame Herba, on rats fed a high-fat diet, Int. J. Obesity, 24, 758-764), Platycodin grandiflorum (Birari RB, Bhutani KK. (2007) Pancreatic lipase inhibitors from natural sources: unexplored potential. Drug Dicov. Today, 12, 879-889). In this study, lipase inhibition ability of native plants was investigated in order to investigate the inhibitory activity of pancreatic lipase from edible plants. Among them, active substances were investigated for the low - Thereby obtaining a substance having excellent activity.

On the other hand, turmeric (薑黄) or 금 金 (九金) is a perennial herbaceous perennial plant belonging to the ginger family (Curcuma longa L.) dried roots origin, India, China, It is cultivated in tropical and subtropical regions mainly in India. It has been known that sexuality is the efficacy of fever, warmth and blood clotting, and transparency, and it is known that it is effective for the treatment of 금 金, 乞 金, (玉 金), kingdom (王金), and turmeric (深 黄) are also called, except in the northern mountainous areas are cultivated in various places. Curcumin, the main ingredient, has been known to have anticancer effects, including anti-inflammatory and antioxidant properties, which cause turmeric to turn yellow. Functional substances in Ulgum (kimchi) are expected to contribute to the prevention of cardiovascular diseases, especially atherosclerosis and hypertension, through mechanisms such as thrombolytic activity, lipid lowering action, LDL oxidation inhibition effect and ACE inhibitory effect However, no disclosure or teaching has been made on the components of the human pacreatic lipase inhibitor from the extract.

Accordingly, the inventors of the present invention have found that, in the process of searching for an active substance that inhibits lipolytic enzymes from natural products, it has been confirmed that diarylheptanoid-based compounds isolated from the genus Ulgum fractions suppress lipolytic enzyme activity, The present invention has been completed.

It is an object of the present invention to provide a pharmaceutical composition for the prevention and treatment of obesity, which comprises a fraction obtained by fractionating a ganoderma lucidum extract with an organic solvent as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating obesity, which comprises a diarylheptanoid compound represented by the following general formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Chemical Formula 1]

(Wherein R is hydrogen or methoxy; and

은 단일 또는 이중결합이다).

Is a single or double bond.

Another object of the present invention is to provide a composition for a health food for preventing and improving obesity, which comprises a fraction obtained by fractionating a ganoderma lucidum extract with an organic solvent as an active ingredient.

Another object of the present invention is to provide a composition for health food for preventing and improving obesity, which comprises a diarylheptanoid compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient .

[Chemical Formula 1]

(Wherein R is hydrogen or methoxy; and

은 단일 또는 이중결합이다).

Is a single or double bond.

In order to solve the above problems, the present invention provides a pharmaceutical composition for the prevention and treatment of obesity, which comprises a fraction obtained by fractionating a Korean ginseng extract with an organic solvent as an active ingredient.

The present invention also provides a pharmaceutical composition for the prevention or treatment of obesity comprising the diarylheptanoid compound represented by the above formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the present invention provides a composition for health food for preventing and improving obesity, which comprises a fraction obtained by fractionating a ganoderma lucidum extract with an organic solvent as an active ingredient.

In addition, the present invention provides a composition for health food for preventing and / or improving obesity comprising the diarylheptanoid compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

Since it was confirmed that the organic solvent fraction isolated from the extract according to the present invention and the diarylheptanoid-based compound separated therefrom were excellent in the ability to inhibit dimethoxycalcumin and curcumin lipase, the diallyl heptanoid Based compounds and organic solvent fractions isolated therefrom containing them are useful for the prevention and treatment of obesity.

FIG. 1 shows the HPLC results (A) and the HPLC results (B) of the four extracts isolated from Ulmus.
FIG. 2 is a graph showing hydrolysis results of bismethoxycalcum in accordance with the concentration of lipase in the ethanol extract of Ulmus.
FIG. 3 is a graph showing the results of hydrolysis according to lipase concentration of curcumin, which is isolated from ethanol extract of Ulmus.
FIG. 4 is a graph showing the results of Lineweaver-Burk plot analysis of lipidase inhibition patterns of bismethoxycalcumin separated from ethanol extract of Ulmus.
FIG. 5 is a graph showing the lipid-degrading activity of bismethoxycalcumin isolated from ethanol extract of Ulmus using Dixon plot. FIG.
FIG. 6 is a graph showing the results of Lineweaver-Burk plot analysis of the lipolytic enzyme inhibitory activity of curcumin, which was isolated from the ethanol extract of Ulvae.
FIG. 7 is a graph showing the results of Dixon plot of the inhibition of lipolytic enzymes of curcumin, which was isolated from the ethanol extract of Ulmus.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for the prevention and treatment of obesity, which comprises a fraction obtained by fractionating an urokinase extract with an organic solvent as an active ingredient.

The term " urcuma " aromatica Salisb . "Is meant to include all organs of natural, hybrids, or variegates, including, for example, roots, branches, stems, leaves and flowers,

In the organic solvent, the organic solvent may be selected from the group consisting of n-hexane, ethyl acetate, and water, but is not limited thereto.

The fraction may be, but is not limited to, ethanolic extract of Ulgiin, which is suspended in water and then added with ethyl acetate or n -hexane.

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

[Chemical Formula 1]

(Wherein R is hydrogen or methoxy; and

은 단일 또는 이중결합이다)

Lt; / RTI > is a single or double bond)

The compound may be a compound represented by any one of the following formulas (2) to (5), but is not limited thereto.

(2)

(3)

[Chemical Formula 4]

[Chemical Formula 5]

In order to prepare the uroguanic acid extracts and fractions, the wort was pulverized and extracted by immersion in ethanol. The extracts were filtered and concentrated under reduced pressure to obtain an ethanol extract. To separate the active substances, the fractions were separated by solvent fractionation with hexane, chloroform, butanol, and water to obtain the respective fractions. pancreatic lipase inhibitor activity, the inhibition activity of ethyl acetate fraction was better than that of ethyl acetate fraction.

To separate physiologically active compounds from E. coli extract, the present inventors diluted ethyl acetate fraction with a normal phase (Silicagel column), a size exclusion column (Sephadex-LH 20), a reverse phase column column) and preparative liquid chromatograph column (Prep-LC) were sequentially carried out in parallel to obtain a total of four active substances (Formulas 2, 3, 4 and 5).

The inventors of the present invention measured the structure, molecular weight, molecular formula, mass spectroscopy, ¹ H-NMR spectrum and ¹³ C-NMR spectrum of the above compounds and found that the physicochemical Properties and chemical structure are as follows.

(2)

Bisdemethoxycurcumin

;

;

(3)

Demethoxycurcumin

;

;

[Chemical Formula 4]

Curcumin

[Chemical Formula 5]

5-hydroxy-1,7-bis (4-hydroxyphenyl) -4,6-heptadiene- -3one)

The inventors of the present invention have found that, in order to confirm the lipase inhibitory effect of the uroguanic acid ethanol extract and the fraction thereof, the enzyme solution obtained by dissolving the human lipolytic enzyme (Sigma, BCR694) in the enzyme buffer was supplemented with the wooly ethanol extract, the acetate fraction and the n-hexane fraction After mixing, the substrate ( p -NPB) was incubated and the absorbance was measured. As a result, the lipase inhibitory activity was higher in the uroguanic acid fractions (see Table 1).

In order to confirm the lipase inhibitory effect of the four physiologically active substances isolated from the Uric acid acetate fraction, the lipase inhibitory effects of the human lipolytic enzyme (Sigma, BCR694) in the enzyme buffer solution were measured, ( P- NPB) was added and incubated, and the absorbance was measured. As a result, lipase inhibitory activity was higher in dimethoxycalcumin (Formula 3) and curcumin (Formula 4) See Table 2).

The inhibitory activity of dimethoxycalcumin (Formula 3) and curcumin (Formula 4), which were separated from the extracts, was determined by the concentration of lipolytic enzyme and the concentration of dimethoxycalcumin (Formula 3) and curcumin (Formula 4) (Fig. 2 and Fig. 3).

In order to investigate the effect of dimethoxycalcumin (3) on the lipolytic enzymes in order to inhibit the activity of dimercoxycalcumin (Formula 3) isolated from the extract, the IC ₅₀ And Ki values and Dixon plots were used to examine competitive and noncompetitive enzyme inhibition patterns. As a result, dimethoxycalcumin (Formula 3) was found to bind to other sites other than the active site of the enzyme, Thereby reducing affinity (Figs. 4 and 5).

In order to investigate the effect of curcumin (Formula 4) isolated from the extract on the lipase to inhibit its activity, curcumin (Formula 4) isolated from the koi extract was subjected to IC ₅₀ And Ki values, and Dixon plots were used to examine competitive and noncompetitive enzyme inhibition patterns. As a result, curcumin (Formula 4) (Fig. 6 and Fig. 7) by binding to another site other than the active site of the enzyme.

Therefore, among the diolheptanoid compounds isolated from the ginseng extract, the fractions thereof and the fraction according to the present invention, dimethoxycalcumin (Formula 3) and curculin (Formula 4) are excellent in lipase inhibitory activity, It was confirmed that the extracts and fractions were twice as excellent as the extracts and fractions thereof, and thus they could be usefully used as a preventive and therapeutic agent for obesity.

The composition of the present invention may contain, as an active ingredient, 0.1 to 99.9% by weight of the herbal composition of the present invention or a fraction thereof, 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, The dose is 0.01 to 1000 mg / kg, preferably 30 to 500 mg / kg, more preferably 50 to 300 mg / kg, and can 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.

In addition, the present invention provides a composition for health food for preventing and improving obesity, which comprises a fraction obtained by fractionating a ganoderma lucidum extract with an organic solvent as an active ingredient.

In the organic solvent, the organic solvent may be selected from the group consisting of n-hexane, ethyl acetate, and water, but is not limited thereto.

The fraction may be, but is not limited to, ethanolic extract of Ulgiin, which is suspended in water and then added with ethyl acetate or n -hexane.

In addition, the present invention provides a composition for health food for prevention and improvement of obesity comprising a diarylheptanoid compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Chemical Formula 1]

(Wherein R is hydrogen or methoxy; and

은 단일 또는 이중결합이다)

Lt; / RTI > is a single or double bond)

The compound may be a compound represented by any one of the following formulas (2) to (5), but is not limited thereto.

(2)

(3)

[Chemical Formula 4]

[Chemical Formula 5]

In the present invention, it has been confirmed that dimethoxycalcumin (Formula 3) and curcumin (Formula 4) are excellent in lipolytic enzyme inhibitory activity, among the diolheptanoid compounds separated from the herbal extracts, fractions thereof and fractions thereof , Dimethoxycalcumin (Formula 3) and curcumin (Formula 4) of the present invention, or a fraction thereof, can be used as an active ingredient of a composition for health food for preventing and improving obesity.

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> Kool ( urcuma aromatica Salisb . ) Preparation of extract

The chrysanthemum used in the present invention was purchased from Hyehwa-dang, Jeongeup city, Jeollabuk-do, Korea. 2 kg of shredded kelp was extracted three times with 18 liters of 95% ethanol for one week at room temperature and then concentrated under reduced pressure to obtain 120 g of ethanol extract.

< Example  2> Kool ( urcuma aromatica Salisb . ) Of the extract Active fraction  Produce

In order to prepare the active fractions of the ganoderma lucidum extract, 120 g of the ethanol extract of Example 1 was suspended in 1 L of distilled water, and the solvent was fractionated with n-hexane, ethyl acetate and water to prepare n- hexane, 24 g of the fraction, 70.4 g of the ethyl acetate fraction and 22 g of the water fraction were obtained.

< Example  3> Kool  Isolation of compounds having physiological activity from the extract

70.4 g of the ethyl acetate fraction of Example 2 was subjected to column chromatography (Sephadex-LH 20), reverse phase column chromatography (C-phase chromatography, -18 column) and a preparative liquid chromatograph column (Prep-LC) were sequentially sequenced to separate and purify a total of four active substances (Formula 2, Formula 3, Formula 4 and Formula 5).

< Example  4> Kool  Identification of compounds isolated from extracts

In order to identify the structure of the purified physiologically active substance, the structure was identified using a spectrometer such as HPLC, Mass and NMR. The purity and retention time of the separated material and the molecular weight of the separated material were confirmed by HPLC. The number of carbon atoms was confirmed by ¹ H-NMR and the number of carbon atoms by ¹³ C-NMR, The diarylheptanoid structure was identified.

As a result, it was found that the compound isolated from the extract was found to be bisdemethoxycurcumin (2), demethoxycurcumin (3), curcumin (4), 5-hydroxy- 5-hydroxy-1,7-bis (4-hydroxyphenyl) -4,6-heptadiene-3one 5) were identified. The physicochemical properties and the structure of the compounds isolated from the ganoderma lucidum extract are as follows.

은 단일 또는 이중결합이다.)
(Wherein R is hydrogen or methoxy; and

Is a single or double bond.)

1) Appearance: Yellow powder

2) Melting point: 222-226 ℃

2) m / z: 208

3) Molecular formula: C ₁₉ H ₁₆ O ₄

^{4) 1 H-NMR (acetone} -d6,500 ㎒) δ 7.61 (d, J = 16.04 Hz 2H), 7.61 (d, J = 8.59 Hz 4H), 6.90 (d, J = 8.59 Hz, 4H), 6.68 -6.65 (d, J = 15.75 Hz 2H), 5.98 (1H).

5) ¹³ C-NMR (acetone-d6, 125 MHz) 183.7, 159.7, 140.2, 160.2, 126.9, 121.24, 116.0, 100.9

1) Appearance: Yellow powder

2) Melting point: 220-225 ℃

3) m / z: 338

4) Molecular formula: C ₂₀ H ₁₈ O ₅

5) ¹ H-NMR (acetone-d 6, 500 MHz)? 7.62-7.55 (4H), 7.34 (1H), 7.18 (1H), 6.89 (3H), 6.70 )

6) ¹³ C-NMR (acetone-d6, 125 MHz)? 56.38, 101.79, 111.53, 116.30, 116.89, 122.12, 122.35, 123.98, 127.77, 128.24, 131.06, 141.13, 141.48, 148.87, 150.13, 160.64, 184.66.

1) Appearance: Yellow powder

2) Melting point: 180-185 ° C

3) m / z: 368.3

4) Molecular formula: C ₂₁ H ₂₀ O ₆

5) ¹ H-NMR (acetone-d 6, 500 MHz) ? 7.62 (2H, d, J = 15.80 Hz, H-4, H- , 6.90 (2H, d, J = 8.3, 1.9 Hz, H-10, H-10 '), 6.83 d, J = 8.15 Hz, H-9, H-6 '), 5.99 (1H, s, H-1), 3.9 (6H).

6) ¹³ C-NMR (acetone-d6, 125 MHz) δ 184.94, 150.44, 149.20, 141.81, 128.58, 124.25, 122.72, 116.64, 111.95, 102.01, 56.72.

1) Appearance: Yellow powder

2) Melting point: 190 ℃

3) m / z: 310

4) Molecular formula: C ₁₉ H ₁₈ O ₄

5) ¹ H-NMR (acetone-d ₆ , 500 MHz)? 7.56-7.52 (2H), 7.06 (2H, d, J = 8.59 Hz), 6.88 d, J = 8.59 Hz), 6.53 (1H, d, J = 15.75 Hz), 5.79 (1H, s). 2.85 (2H, t, J = 7.16 Hz), 2.67 (1H, t, J = 7.16 Hz).

6) ¹³ C-NMR (acetone- d ₆ , 125 MHz)? 200.3, 179.1, 160.5, 156.7, 140.6, 132.7, 130.9, 130.2, 127.8, 120.8, 116.9, 116.1, 101.0, 42.7, 31.2.

< Example  5> Kool  Analysis of Active Substances of Extracts and Compounds Isolated therefrom

The active material was analyzed for profiling of the ugly ethanol extracts and the diarlyheptanoid-based compounds isolated therefrom.

Specifically, a reverse phase column (Agilent, RP-18, Bonus-RP, 5 mm, 150 * 4.6 mm) and 254 nm UV absorbance were added to high performance liquid chromatography (HPLC, Agilent 1200 series) The ethanol extract and the separated active substance were analyzed with a gradient condition of acetonitrile at a flow rate of 0.5 ml / min. The retention time of the active substance and the retention time peaks of the extract were compared with each other Respectively. Gradient conditions were acetonitrile 5 - 25% from 0 min to 10 min, acetonitrile 25 - 45% from 10 min to 20 min, acetonitrile 45 - 65% from 20 min to 30 min, , Acetonitrile 65% - 100% for 30 minutes to 40 minutes, and 100% - 100% for 40 minutes to 50 minutes. (Fig. 1, peak 1) at 39.217 min, demethoxycurcumin (Fig. 1, peak 2) at 37.337 min and curcumin Hydroxy-1, 7-bis (4-hydroxyphenyl) -4,6-heptadien- 4 peak was observed (Figure 1, peak 4), and the retention time of each of the four active substances was compared with the retention time of the ethanol extract To profiling the four active substances contained in the ethanol extract.

< Experimental Example  1> Kool  Activity isolated from the extract Fraction  Measurement of lipase inhibitory activity

(Sigma, BCR694) and p- nitrophenyl butyrate ( p- NPB) were used as substrates in order to confirm the lipase inhibitory effect of the extracts and fractions of Uric acid ethanol.

Specifically, the human lipolytic enzyme is dissolved in an enzyme buffer (10 mM MOPS, 1 mM EDTA, pH 6.8) to prepare an enzyme solution. The extract is dissolved in DMSO to a final concentration of 1 mM and diluted to various concentrations. Substrate solution as is used for the reaction by 50 μL 420 μM are presented after the p -NPB dissolved in DMF. First, 10 μL of the enzyme solution except for the substrate and 50 μL of 0.1 M Tris-HCl buffer (pH 8.2) were mixed. After the addition of the extract, shaking incubation was performed at 37 ° C. for 15 minutes, After shaking incubation for 30 minutes at 40 ° C, the absorbance was measured using a microplate absorbance reader (Biorad 680) at 405 nm. The lipolytic enzyme inhibitory activity was expressed by the absorbance reduction rate of the sample solution added group and the no-added group, and p- nitrophenol was used as the standard material.

The extracts were repeatedly extracted with ethanol, and the extracts were suspended in water and extracted with organic solvent using ethylacetate and n - hexane. The lipase inhibitory activity of each extract was measured.

As a result, it was confirmed that the ethanol extract had a degree of inhibition of 82.2%, the ethyl acetate fraction was 99.4%, and the n-hexane fraction was 40.9% at a concentration of 250 μg / ml.

Ulgum  Ethanol extract and From the fraction  Analysis of lipase inhibitory activity density
(μg / ml) Inhibitory activity (%) IC ₅₀
(μg / ml) 250 125 62.5 31.3 15.6 Ethanol extract 82.2 76.6 55.2 21.2 17.4 59.5 Ethyl acetate fraction layer 99.4 91.6 78.9 49.7 28.0 29.5 n -hexane fraction layer 4.09 25.7 11.7 6.6 2.9 > 250

< Experimental Example  2> Kool From the fraction  Measurement of lipase-inhibiting activity of isolated compounds

(Sigma, BCR694) and p- nitrophenyl butyrate ( p- NPB) were used as substrates in order to confirm the lipase inhibitory effect of the compounds isolated from the Uric acid ethanol extract.

Specifically, the human lipolytic enzyme is dissolved in an enzyme buffer (10 mM MOPS, 1 mM EDTA, pH 6.8) to prepare an enzyme solution. Four active compounds isolated from the ethyl acetate layer fraction are dissolved in DMSO to a final concentration of 1 mM and diluted to various concentrations. Substrate solution as is used for the reaction by 50 μL 420 μM are presented after the p -NPB dissolved in DMF. First, 10 μL of the enzyme solution except for the substrate and 50 μL of 0.1 M Tris-HCl buffer (pH 8.2) were mixed, and 4 active substances isolated from the ethyl acetate layer fraction were added thereto. After shaking incubation for 15 minutes, the substrate was added and shaking incubation was performed at 37 ° C for 30 minutes. Absorbance was measured at 405 nm using a microplate absorbance reader (Biorad 680). The lipolytic enzyme inhibitory activity was expressed by the absorbance reduction rate of the sample solution added group and the no-added group, and p- nitrophenol was used as the standard material.

As a result, it was found that 26.6% of bisdemethoxycurcumin, 95.4% of demethoxycurcumin and 4% of curcumin were active agents at 200 μM of the active substance isolated from the ethyl acetate layer fraction, 76.6%, and 5% (5-hydroxy-1,7-bis (4-hydroxyphenyl) -4,6-heptadiene-3one) of 10.1%. Specifically, dimethoxycalcumin , And curcumin (Formula 4) have IC ₅₀ values of 57.7 μM and 71.4 μM, respectively, which are higher than those of other substances, and are shown in Table 2 below.

Kool Analysis of lipase inhibitory activity from compounds isolated from extracts Inhibitor Inhibition rate ^a (%) IC ₅₀ ^b
(μM) IC ₅₀ ^b
(μg / ml) Bismethoxycalcumin (Formula 2) 26.6 ND ^c ND ^c Dimethoxycalcumin (Formula 3) 95.4 57.7 19.8 Curculin (Formula 4) 76.6 71.4 26.3 Hydroxy-1,7-bis (4-hydroxyphenyl) -4,6-heptadien-3-one
(Formula 5) 10.1 ND ^c ND ^c

^a) inhibition rate at a concentration of 200 [mu] M

^b) All experiments were repeated with 3 replicates.

^c) ND = not measured.

< Experimental Example  3> Kool  extract from  Measurement of inhibitory activity of isolated compounds according to lipidolytic enzyme concentration

In order to determine the inhibitory activity of dimethoxycalcumin (Formula 3) and curcumin (Formula 4) Human lipolytic enzymes (Sigma, BCR694) and p- nitrophenyl butyrate ( p- NPB) were used as substrates and were performed as follows.

Specifically, the human lipolytic enzyme is dissolved in an enzyme buffer (10 mM MOPS, 1 mM EDTA, pH 6.8) to prepare an enzyme solution. The extract is dissolved in DMSO to a final concentration of 1 mM and diluted to various concentrations. Substrate solution as is used for the reaction by 50 μL 420 μM are presented after the p -NPB dissolved in DMF. First, 10 μL of the enzyme solution except the substrate and 50 μL of 0.1 M Tris-HCl buffer (pH 8.2) were mixed. Dimethoxycalcumin and curcumin were added to the extract, and shaking incubation was performed at 37 ° C. for 15 minutes. (shaking incubation), followed by shaking incubation at 37 ° C for 30 minutes. Subsequently, the absorbance was measured at 405 nm using a microplate absorbance reader (Biorad 680).

As a result, it was confirmed that both the dimethoxycalcumin (Formula 3) and curcumin (Formula 4) significantly lowered the residual activity of the lipolytic enzyme as the concentration increased (FIGS. 2 and 3).

< Experimental Example  4> Kool  Extracted from the extract Dimethoxycalcumin (Formula 3)  Lipase inhibition assay

In order to investigate the effect of dimethoxycalcumin (3) on the lipolytic enzyme to inhibit its activity, the dimethoxycalcumin (3) isolated from the uroepyme extract was subjected to Lineweaver-Burk plot And Dixon plot.

Specifically, the human lipolytic enzyme is dissolved in an enzyme buffer (10 mM MOPS, 1 mM EDTA, pH 6.8) to prepare an enzyme solution. The extract is dissolved in DMSO to a final concentration of 1 mM and diluted to various concentrations. Substrate solution as is used for the reaction by 50 μL 420 μM are presented after the p -NPB dissolved in DMF. First, 1.25 μL of the enzyme solution except for the substrate was mixed with 50 μL of 0.1 M Tris-HCl buffer (pH 8.2), and then the extract was added and shaking incubation was performed at 37 ° C. for 15 minutes The following substrates were added and shaking incubated at 37 ° C for 30 min. Then, a Lineweaver-Burk plot was generated using absorbance values at 405 nm using a microplate absorbance reader (Biorad 680). For the Dixon plot, p- NPB should be dissolved in DMF at 420, 210, and 105 μM as a substrate solution. v Firstly, 10 μL of enzyme solution and 50 μL of 0.1 M Tris-HCl buffer (pH 8.2) except for the substrate were mixed. After shaking incubation at 37 ° C for 15 minutes, The substrate was incubated at 37 ° C for 30 min with shaking, and the absorbance was measured at 405 nm using a microplate absorbance reader (Biorad 680).

As a result, the value of dimethoxycalcumin (Formula 3) was found to have a different value of V _max (μM / min) according to each concentration through the value shown in the Lineweaver-Burk plot, the non-competitive was confirmed that the inhibition reaction (noncompetitive inhibition), creating results Ki values of each x-axis point of the Ki value graph meet the different concentration of the Dixon plot for reducing the affinity of the enzyme to the substrate is a 56.7 μM (Figs. 4 and 5).

< Experimental Example  5> Kool  Extracted from the extract Curcumin (Formula 4)  Lipase inhibition assay

A Lineweaver-Burk plot and a Dixon plot were performed on curcumin (Formula 4) isolated from Korean cedarwood extract to investigate how curcumin (Formula 4) isolated from cucurbitaceae extract inhibits activity by acting on lipolytic enzyme Respectively.

Specifically, the IC ₅₀ for lipolytic enzymes And Ki values were obtained and Dixon plots were drawn to examine competitive and noncompetitive enzyme inhibition patterns.

The human lipolytic enzyme is dissolved in an enzyme buffer (10 mM MOPS, 1 mM EDTA, pH 6.8) to prepare an enzyme solution. The extract is dissolved in DMSO to a final concentration of 1 mM and diluted to various concentrations. Substrate solution as is used for the reaction by 50 μL 420 μM are presented after the p -NPB dissolved in DMF. First, 1.25 μL of the enzyme solution except for the substrate was mixed with 50 μL of 0.1 M Tris-HCl buffer (pH 8.2), and then the extract was added and shaking incubation was performed at 37 ° C. for 15 minutes The following substrates were added and shaking incubated at 37 ° C for 30 min. Then, a Lineweaver-Burk plot was generated using absorbance values at 405 nm using a microplate absorbance reader (Biorad 680). For the Dixon plot, p- NPB should be dissolved in DMF at 420, 210, and 105 μM as a substrate solution. First, 10 μL of the enzyme solution except for the substrate and 50 μL of 0.1 M Tris-HCl buffer (pH 8.2) were mixed. The extract was added and shaking incubation was performed at 37 ° C. for 15 minutes. Subsequently, And incubated at 37 ° C for 30 min. After shaking incubation, absorbance was measured at 405 nm using a microplate absorbance reader (Biorad 680).

As a result, through the values shown in the Lineweaver-Burk plot, curcumin (Formula 4) It is confirmed that the enzyme has a different V _max (? / Min) value according to each concentration, and it is a noncompetitive inhibition reaction which binds to a site other than the active site of the enzyme and reduces the affinity of the substrate for the enzyme could find, create results Ki values of each x-axis point of the Ki value of the graph is to see different levels of the Dixon plot was confirmed to exhibit a 73.9 μM (Fig 6 and 7).

< Manufacturing example  1> Sanje  Produce

0.1 g of bismethoxycalc citric acid isolated from the koi extract of Example 4

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 > g &lt; / RTI &gt; of dimethoxy curcumin

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

0.1 g < RTI ID = 0.0 > of curcumin &lt; / RTI &gt;

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

Hydroxy-1,7-bis (4-hydroxyphenyl) -4,6-heptadiene-3-one obtained in Example 4

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 < RTI ID = 0.0 > of curcumin &lt; / RTI &gt;

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 curcumin, which was isolated from the koji extract of Example 4

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 curcumin, which was isolated from the koji extract of Example 4

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 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 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 (10)

A pharmaceutical composition for the prevention and treatment of obesity, which comprises a fraction obtained by fractionating corynebacterium extract with an organic solvent as an active ingredient.
The pharmaceutical composition for preventing and treating obesity according to claim 1, wherein the organic solvent is selected from the group consisting of n-hexane, ethyl acetate and water.
The pharmaceutical composition for the prevention and treatment of obesity according to claim 1, wherein the fraction is a soluble fraction separated from water after the ethanol extract of cowpea is suspended in water and then ethyl acetate or n -hexane is added thereto.
1. A pharmaceutical composition for preventing or treating obesity comprising as an active ingredient a diarylheptanoid compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof:
[Chemical Formula 1]

(Wherein R is hydrogen or methoxy; and

Is a single or double bond.
5. The method of claim 4,
The pharmaceutical composition for preventing or treating obesity is characterized in that the compound is a compound represented by any one of the following formulas (2) to (5)
(2)

;
(3)

;
[Chemical Formula 4]
; And

[Chemical Formula 5]

.
A composition for health food for the prevention and improvement of obesity, which comprises a fraction obtained by fractionating an uleguk extract with an organic solvent as an active ingredient.
The composition according to claim 6, wherein the organic solvent is selected from the group consisting of n-hexane, ethyl acetate and water.
[Claim 7] The composition according to claim 6, wherein the fraction is a soluble portion separated from water after the ethanol extract of Ulgum is suspended in water and then ethyl acetate or n -hexane is added thereto.
A composition for health food for prevention and improvement of obesity comprising diarylheptanoid compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:

[Chemical Formula 1]

(Wherein R is hydrogen or methoxy; and

Is a single or double bond.
10. The method of claim 9,
Wherein the compound is a compound represented by any one of the following formulas (2) to (5):
(2)

;
(3)

;
[Chemical Formula 4]

; And

[Chemical Formula 5]

.

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