KR101014004B1 - Compositions and functional foods for the treatment and prevention of obesity - Google Patents

Abstract

The present invention relates to the ethyl acetate layer of the extract of T. aeruginosa and 3 β ( trans - p- coumaroyl) oxy-2 a , 23-dihydroxyurs-12: 20 (30) -dien-28-oic acid [ It was confirmed that 3- O - trans - p- coumaroyl actinidic acid] effectively inhibited the efficacy of pancreatic lipase, one of the causes of obesity.

Thus, the ethyl acetate layer of the Tara root extract of the present invention and 3 β ( trans - p- coumaroyl) oxy-2 a , 23-dihydroxyurs-12: 20 (30) -dien-28-oic, which is a novel compound separated therefrom Acid [3- O - trans - p -coumaroyl actinidic acid] may be useful in pharmaceutical composition for preventing, treating or ameliorating obesity, and in dietary supplements.

Sputum, pancreas, lipase, obesity,

Description

Compositions and functional food for prevention and treatment of obesity

The present invention is a novel compound isolated from the future 3 β ( trans - p -coumaroyl) oxy-2 a , 23-dihydroxyurs-12: 20 (30) -dien-28-oic acid [3- O - trans - p- coumaroyl actinidic acid] and a pharmaceutical composition for preventing, treating or improving obesity using the same, and a health functional food.

Obesity is caused by an imbalance in energy absorption and consumption. The extra energy is stored in fat cells, increasing the number or size of fat cells. It is also one of the strongest risk factors for various diseases, including hypertension, hyperlipidemia, arteriosclerosis, and diabetes (Borgstrom, B., Exocrine Pancreas, Pathology and Diseases.Raven Press, New York, pp. 361-401, 1981). As such, many studies are being conducted to treat obesity, which is the source of all diseases. Many studies are underway to treat obesity. One of the therapies to prevent and treat obesity is to reduce the absorption of fatty acids by inhibiting the effect of lipase in the digestive tract (Ballinger, A., et al., Eur . J. Pharm . Sci ., 440, 109-117). , 2002; Yanovski, SJ, et al., New Engl . J. Med ., 346, 591-602, 2002). Pancreatic lipase is a key enzyme that enhances fat absorption by catalyzing the hydrolysis of triacylglycerol into 2-monoacylglycerol and fatty acids in the gastrointestinal tract (Bitou, N., et al., Lipids , 34, 441-445, 1999). Therefore, effective and specific inhibition of this enzyme has been reported to have the effect of preventing, treating or ameliorating obesity (Gargouri, Y., et al., Biochim . Biophys . Acta , 1344, 6-37, 1997). ).

A representative pancreatic lipase inhibitor currently known is tetrahydrolipstatin (Orlistat), a derivative of lipstatin derived from Streptomyces toxitricini , which was approved by the US FDA as a drug for long-term administration and commercialized under the trade name Genical. This is the most sold in the world. Xenical is known to be the most potent to inhibit about 30% of the fat consumed (Drent, ML, et al., Int . J. Obes ., 19, 221-226 , 1995). By inactivating an irreversible bond to the lipase that degrades triglycerides, it reduces triglyceride absorption and further cholesterol absorption. Triglycerides and cholesterol that are not degraded are unconverted and excreted in feces.

However, even long-term administration (two years) in clinical practice has not been reported to be superior to the diet alone. In addition, there are known side effects such as facial paralysis, gastrointestinal disorders, hypersensitivity, bile secretion disorders, and fat-soluble vitamin absorption inhibition (Drent, ML, et al., Int . J. Obes ., 17, 241-244, 1993; Peter, C., et al., Br . J. Clin . Pharmacol ., 51, 135-141, 2001).

In Korea, 30.4% (241/793) of adverse reactions were reported as a result of post-marketing results survey of 793 people for review. In particular, 29.9% (237 persons) were causally related to Jennal among the adverse reactions. The most common adverse reactions were gastrointestinal symptoms (29%, 230 patients). Among them, new adverse reactions that did not occur in pre-clinical studies were skin sensation, constipation, indigestion, and heartburn. In particular, the FDA warned that excessive weight loss could increase the risk of cholelithiasis through a change in permit. In clinical trials for the prevention of type 2 diabetes, the incidence of gallstones was reported to be 2.9% (47/1649) in the drug group and 1.8% (30/1655) in the placebo group (Daily Farm, Oct. 02, 2007). In addition, it has been reported that a lot of inconvenience to the life while taking diarrhea with practically unpredictable.

In order to solve the above problems, 3 β ( trans - p- coumaroyl) oxy-2, which is a novel compound separated from the ethyl acetate layer fraction and the ethyl acetate layer fraction, has been shown to be effective in inhibiting lipase in digestion inhibitors. a , 23-dihydroxyurs-12: 20 (30) -dien-28-oic acid [3- O - trans - p- coumaroyl actinidic acid] to provide a pharmaceutical composition, health functional food for the prevention, treatment or improvement of obesity The purpose is to provide.

In order to achieve the object of the present invention, the root of the stalk according to the present invention is chopped and extracted with an alcohol or an aqueous alcohol solution, and then separated into a system fractionation layer, an ethyl acetate layer fraction, and an ethyl acetate layer of the root extract. A new compound, 3 β ( trans - p -coumaroyl) oxy-2 a , 23-dihydroxyurs-12: 20 (30) -dien-28-oic acid [3- O - trans - p -coumaroyl actinidic acid] It is characterized by the prevention, treatment or improvement of the pharmaceutical composition, health functional food containing.

Ethyl acetate layer fraction of the root extract of the present invention and 3 β ( trans - p -coumaroyl) oxy-2 a , 23-dihydroxyurs-12: 20 (a new compound isolated from the ethyl acetate layer of the root extract of 30) -dien-28-oic acid [3- O - trans - p- coumaroyl actinidic acid] was found to effectively inhibit the efficacy of pancreatic lipase, one of the causes of obesity.

As a representative obesity treatment agent, Zenical is causing fatal inconveniences in daily life due to poor efficacy and unpredictable diarrhea, it has not only a new material development but also a domestic product that can cope with FTA. Considering the inevitable, the ethyl acetate layer fraction of the root extract of the present invention and the novel compound may be used as a material for the prevention, treatment or improvement of obesity or related diseases.

The present invention relates to 3 β ( trans - p- coumaroyl) oxy-2 a , 23-dihydroxyurs-12: 20 (30) -dien-28-oic acid isolated from the above [3- O - trans - p- coumaroyl actinidic acid It is related with ethyl acetate layer fraction which is a phylogenetic layer by extracting a novel compound having a molecular formula of] and the root of the stalk with an alcohol or an aqueous alcohol solution, and then systematically separating it with hexane, ethyl acetate, butanol and water.

[[ Actinidia arguta Planchon; Actinidiaceae] is a type of kiwifruit with a soft shell and is native to Korea, China, Japan and Siberia (Ferguson, R., New). Zealand Kiwifruit , 81, 23-24, 1991). Darae has been used not only as a food for a long time, but also as a traditional medicine for improving general health (Kim, J.-G., et al., Traditional Drugs of the East Color Edition.Young-Rim Publishing Co., Seoul, p. 207, 1995). Studies on the constituents of the fungus include flavonoids (Webby, RF, et al., Phytochemistry , 29, 289-292, 1990), phenolic compounds (Lim, H.-W., et al., Nat . Prod . Sci . , 12, 221-225, 2006), triterpene and lignan (Whang, JI, et al., Kor . J. Pharmacogn ., 31, 357-363, 2000). But there are no reports of obesity.

Hereinafter, the present invention will be described in detail with reference to Examples.

3 β ( trans - p- coumaroyl) oxy-2 a , 23-dihydroxyurs-12: 20 (30) -dien-28-oic acid [3- O - trans - p -coumaroyl actinidic acid, a novel compound according to the present invention ] Has the following formula.

New compound of formula 1: colorless crystals (MeOH). mp 265-267 ° C. [a] _D ²⁵ + 84.6 ° ( c 0.20, pyridine);

UV (MeOH) λ _max (log ε): 312 (4.37) nm; IR _max (NaCl) 3490-3380, 3086-2850, 1712, 1667, 1631, 1600, 1515, 1450, 1331, 1291, 1192, 1167, 1023, 959, 828, 656 cm ⁻¹ ;

¹ H NMR (300 MHz, pyridine- d ₆ ) δ 7.97 (1H, d, J = 16.0, H-7 '), 7.56 (1H, d, J = 9.0, H-2' / 6 '), 7.17 ( 1H, d, J = 8.5, H-3 '/ 5'), 6.65 (1H, d, J = 16.0, H-8 '), 5.92 (1H, d, J = 9.5, H-3), 5.47 ( 1H, br t, H-12), 4.82 (1H, br s, H-30a), 4.77 (1H, br s, H-30b), 4.46 (1H, ddd, J = 11.0, 9.5, 4.5, H- 2), 3.64 (1H, d, J = 11.5, H-23a), 3.50 (1H, d, J = 11.5, H-23b), 2.75 (1H, d, J = 11.5, H-18), 2.36 ( 1H, dt, J = 12.5, 4.5, H-1a), 1.85 (1H, dd, J = 10.0, 7.0, H-7 '), 1.44 (1H, brt, J = 12.0, H-1b), 1.17 (3H, s, H-27), 1.10 (3H, d, J = 6.5, H-29), 1.08 (3H, s, H-25), 1.07 (3H, s, H-26), 0.96 (3H , s, H-24);

¹³ C-NMR (75 MHz, pyridine- d ₆ ) δ 179.6 (C-28), 168.8 (C-9 '), 161.8 (C-4'), 154.1 (C-20), 145.4 (C-8 ' ), 139.4 (C-13), 131.0 (C-2 '/ 6'), 126.6 (C-1 '), 126.2 (C-12), 117.2 (C-3' / 5 '), 116.2 (C- 7 '), 105.5 (C-30), 80.3 (C-3), 67.0 (C-2), 65.2 (C-23), 56.0 (C-18), 48.9 (C-1), 48.6 (C- 17), 48.3 (C-5), 47.6 (C-9), 44.2 (C-4), 43.0 (C-14), 40.4 (C-8), 40.0 (C-22), 38.4 (C-10 ), 38.1 (C-19), 33.4 (C-7), 33.1 (C-21), 29.0 (C-15), 25.2 (C-16), 24.12 (C-11), 24.07 (C-27) , 18.6 (C-6), 17.83 (C-25), 17.80 (C-26), 16.9 (C-29), 15.2 (C-24); EIMS m / z (rel. Int.) 632 ([M] ⁺ , 3), 485 (2), 387 (10), 246 (33), 201 (56), 187 (17), 147 (100), 119 (39);

HREIMS m / z : 632.3710 ([M] ⁺ , Calcd for C ₃₉ H ₅₂ O ₇ : 632.3713).

The novel compounds according to the present invention were obtained as colorless crystals and obtained by molecular ionization by HREIMS.m / z 632.3710 [M]⁺Was obtained, which is the elemental formula C₃₉H₅₂O₇To match. In the EIMS spectrum of the new compoundmOfz Fragment ions of 246 and 201 (246-COOH) were observed, which can be explained by the retro-Diels-Alder cleavage typical of urs-12-ene with carboxyl groups (Aquino, R., De Simone, F., Vincieri, FF, Pizza, C., Gacs-Baitz, E., New polyhydroxylated triterpenes fromUncaria tomentosa.J. Nat . Prod53, 559-564, 1990; Budzikiewicz, H., Wilson, J. M., Djerassi, C., Mass spectrometry in structural and stereochemical problems. XXXII. Pentacyclic triterpenes.J. Am. Chem . Soc, 85, 3688-3699, 1963). In the IR spectrum, hydroxyl (3490-3380 cm)^-One), conjugated ester (1712 cm^-One) And carbonyl (1667 cm^-One) Absorption was observed. Previously except that an exocyclic methylene group was observed instead of a methyl group in the E ring.Actinidia polygama3-, coumaroyl urs-12-ene isolated fromO-trans-p-coumaroyl asiatic acid (Sashida, Y., Ogawa, K., Mori, N., Yamanouchi, T., Triterpenoids from the fruit galls ofActinidia polygama.Phytochemistry, 31, 2801-2904, 1992) and the novel compounds^OneH and¹³C chemical shift values were very similar.trans-pThe presence of -Coumaroyl groupδ _H 6.65 (1 H, d,J = 16.0 Hz), 7.17 (2H, d,J = 8.5 Hz), 7.56 (2H, d,J = 9.0 Hz) and 7.97 (1 H, d,J = 16.0 Hz)^OneH-NMR resonance and EIMS in the spectrummOfz 147 could be expected as the major fragment ion peak.

Four tertiary methyl groups ( δ _H 1.17, ¹ H-NMR (FIG. 1) and COSY (FIG. 4) were selected correlations of the new compounds with the selected correlations observed in COSY (━) and HMBC (→) NMR spectra. 1.08, 1.07 and 0.96, respectively 3H), one secondary methyl group ( d _H 1.10, J = 6.5 Hz, 3H), one trisubstituted olefinic double bond ( d _H 5.47, br t) and one The exocyclic methylene group ( δ _H 4.82 and 4.77, each br s) was confirmed.

In addition, two oxymethine protons resonating at δ _H 5.92 (d, J = 9.5 Hz, 1H) and 4.46 (ddd, J = 11.0, 9.5, 4.5 Hz, 1H) and δ _H 3.64 and 3.50 (d, J = Hydroxymethyl protons resonating at 11.5 Hz, 1H) were observed in the ¹ H-NMR spectrum of the novel compounds. ¹³ C-NMR (FIG. 2) and DEPT 135 spectra (FIG. 3) of the novel compounds also confirmed the presence of exocyclic methylene groups at δ _C 105.5 and 154.1. The positions of all substituents present in Compound 1, including O - trans - p- Coumaroyl (C-3), exocyclic methylene group (C-20), were represented by COSY (FIG. 4), HMQC (FIG. 5) and HMBC NMR (FIG. 6). ) Through the technique. The coupling constant ( J = 9.5 Hz) between the H-2 α and H-3 β protons observed in the ¹ H-NMR spectrum of the novel compound was related to 3- O - trans - p- coumaroyl asiatic acid ( J = 9.8 Hz). ) (Sashida et al ., 1992) or actinidic acid ( J = 9.6 Hz) (Lahlou, EH, Hirai, N., Kamo, T., Tsuda, M., Ohigashi, H., Actinidic acid, a new triterpene phytoalexin from unripe kiwi fruit . Biosci. Biotechnol. Biochem., by the fact that similar to 65, 480-483, 2001) were able to derive a 2 α, 3 β configuration of the two secondary hydroxyl groups present in the novel compounds.

This prediction was confirmed through the NOESY spectrum of the new compound (FIG. 7). In Chemical Formula 3, NOE was observed between H-2 and H-25 as selected correlations observed in the NOESY (↔) NMR spectrum of the novel compound, which means that H-2 and H-3 are at β and α positions, respectively. do. In addition, strong NOE correlation was observed in H-3 and Hb-23 and H-24 and H-2 / H-25 in the NOESY spectrum of the new compound, and the position of the hydroxymethyl group was determined as C-23.

Thus, the chemical structure of the novel compound of new material 3 β (trans - p -coumaroyl) oxy-2 a, 23-dihydroxyurs-12: 20 (30) -dien-28-oic acid [3- O - trans - p -coumaroyl actinidic acid]. The Actinidic acid mature kiwi fruit (Actinidia deliciosa) separated and was reported (Lahlou, EH, Hirai, N. , Kamo, T., Tsuda, M., Ohigashi, H., Actinidic acid, a new triterpene phytoalexin from unripe kiwi fruit. Biosci. Biotechnol. Biochem from. , 65, 480-483, 2001), p- coumaroyl esters of actinidic acid are the first to be isolated and reported from natural sources.

Ⅰ. Ethanol Extracts of T. Root and Preparation of Effective Novel Compounds

Tara used in the experiment ( Actinidia arguta The roots of the Planchon were collected in April 2006 from Palbongsan, Palbong-myeon, Seosan-si, Chungcheongnam-do, Korea. The specimen of evidence (no. KIOM-ACAR1) is being stored in the herbarium of the Herbal Medicine Research Department of the Korean Institute of Oriental Medicine.

1) Extraction and separation of new material

16 kg of dried and fine stalk root was extracted with 36 L of ethanol for 3 times at room temperature, and then concentrated under reduced pressure at 40 ° C. to obtain an ethanol extract (553 g). Some of the ethanol extract (300 g) was suspended in distilled water (2 L), and then the solvent was fractionated sequentially with normal hexane ( n- hexane, 6 L), ethyl acetate (EtOAc, 6 L) and butanol (BuOH, 6 L). A fraction (50.5 g), an ethyl acetate layer fraction (60.4 g), a butanol layer fraction (73.9 g) and a water layer fraction (115.2 g) were obtained, respectively.

Column chromatography on ethyl acetate layer fraction (50 g) with silica gel ( φ 6.5x47 cm, 70-230 mesh) as fixed phase and CHCl ₃ -MeOH mixed solvent (20: 1 → 0: 1 v / v) as mobile phase Column chromatography was performed and divided into nine fractions (F01-F09). Eluted with fraction F03 [CHCl ₃ -MeOH (8: 1 v / v); 6.28 g] were further subdivided into 10 subfractions (F0301-F0310) by silica gel column chromatography ( φ 6.5x38 cm, 230-400 mesh; CHCl ₃ -MeOH 20: 1 → 1: 1 v / v). . A novel compound (82 mg) represented by Chemical Formula 1 was isolated from small fraction F0301 by recrystallization with methanol.

II. Pancreatic Lipase Inhibitory Effect Analysis

(1) Pancreatic Lipolytic Inhibition Experiment

30 μl (10 units) of porcine pancreatic lipase (Sigma, St. Louis, MO, USA) mixed with Enzyme buffer (10 mM MOPS (morpholinepropanesulphonic acid) and 1 mM EDTA, pH 6.8) and Tris buffer (100 mM Tris-HC1) And 5 mM CaCl ₂ , pH 7.0) 850 μl were mixed. Here, 100 μl of various concentrations of the test substance and a control group, Genical (Orlistat; Roche, Basel, Switzerland) were mixed and incubated at 37 ° C. for 15 minutes. Subsequently, 20 μl of a substrate solution [10 mM p- NPB ( p- nitrophenylbutyrate) in dimethyl formamide] was added thereto, followed by further incubation at 37 ° C. for 15 minutes. The p -nitrophenol which is hydrolyzed from p -NPB generated by the enzyme reaction using the ELISA reader (BIO-TEK, Synergy HT, USA) , were measured at the 400 nm wavelength. The degree of inhibition of lipase activity was converted into percent value (%).

(2) Pancreatic Lipolytic Inhibition Test Results

Table 1 was analyzed pancreatic lipase inhibitory effect of the root extract, hexane layer, ethyl acetate layer, butanol layer, water layer and the novel compounds of the present invention. The ethyl acetate layer and the new material are weaker than Xenical [IC ₅₀ value 0.16 μ g / ml (0.32 μ M)], but the IC ₅₀ values are 74.94 μ g / ml and 9.45 μ g / ml (14.95 μ M), respectively. Lipolytic activity was significantly inhibited (see Table 1). Because Xenical causes fatal inconvenience to daily life due to lack of efficacy and unexpected diarrhea,

Ethyl acetate layer of the root of the larvae and a new compound isolated therefrom 3 β ( trans - p- coumaroyl) oxy-2 a , 23-dihydroxyurs- 12:20 (30) -dien-28-oic acid [3- O - trans - p -coumaroyl actinidic acid] could be used as the material for the prevention, treatment or amelioration of obesity and related diseases.

Compound
Inhibitory effect (IC ₅₀ value) ^a) μ g / ml μ M Ethanol extract > 100 . Hexane layer > 100 . Ethyl acetate layer 74.94 . Butanol layer > 100 . Water layer > 100 . New compound 9.45 ± 0.13 14.95 ± 0.21 Xenical (Yangseong Control) 0.16 ± 0.005 0.32 ± 0.01

^a) The concentration of compound required to inhibit pancreatic adipose activity by 50% (IC ₅₀ ). IC ₅₀ values were calculated from dose inhibition curves. Inhibitory effect was expressed as mean ± SD of 4 replicate data.

1 is a ¹ H-NMR spectrum of the compound of Formula 1. FIG.

2 is a ³ C-NMR spectrum of the compound of Formula 1. FIG.

3 is a DEPT-135 NMR spectrum for the compound of Formula 1. FIG.

4 is a ¹ H- ¹ H COSY NMR spectrum for the compound of Formula 1. FIG.

FIG. 5 is a ¹³ C- ¹ H HMQC NMR spectrum for a compound of Formula 1. FIG.

FIG. 6 is a ¹³ C- ¹ H HMBC NMR spectrum for a compound of Formula 1.

7 is a NOESY NMR spectrum for the compound of Formula 1. FIG.

Claims (7)

New compounds represented by the following formula (1): [Formula 1]

The method of claim 1, The compound is characterized in that obtained by extractive separation from the tea. Extracting dried and fine stalks with an alcohol or an aqueous alcohol solution to prepare a stalk extract; Suspending by adding water to the above extract, and dividing into normal hexane, ethyl acetate, butanol to obtain an ethyl acetate layer fraction fractionated with ethyl acetate; Separation and purification of the ethyl acetate layer fractions by chromatography. Separation method of a compound represented by the following formula (1) comprising the step of purifying. [Formula 1]

A pharmaceutical composition for preventing or treating obesity, comprising the compound of claim 1 as an active ingredient. Obesity prevention and improvement health functional food containing the compound of claim 1 as an active ingredient. After extracting the roots of the stalks with an alcohol or an aqueous alcohol solution, the extracted extract was filtered and concentrated under reduced pressure. A pharmaceutical composition for the prevention or treatment of obesity, containing. After extracting the roots of the stalks with an alcohol or an aqueous alcohol solution, the extracted extract was filtered and concentrated under reduced pressure. As a dietary supplement for the prevention and improvement of obesity.

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