KR101132174B1 - AMPK activators from Erythrina abyssinica, and compositions for prevention and treatment of metabolic syndromes through activation of AMPK enzyme containing the same as an active ingredients - Google Patents

Abstract

The present invention is an African plant Erythrina Abyssinica ( Erythrina). Abyssinica ) relates to a compound for activating AMP-activated protein kinase (AMPK) derived from abyssinica and a composition comprising the same as an active ingredient, the composition has a prevention and treatment effect of diabetes, obesity or metabolic syndrome .

Description

AMPK activators from Erythrina abyssinica, and compositions for prevention and treatment of metabolic syndromes through activation of AMPK enzyme containing the same as an active ingredients}

The present invention is an African plant Erythrina Abyssinica ( Erythrina). It relates to a compound for activating AMP-activated protein kinase (AMPK) derived from abyssinica ) and a composition for preventing and treating diabetes, obesity or metabolic syndrome comprising the same as an active ingredient.

More specifically, the present invention is a diabetic, obesity or metabolic syndrome containing an AMPK activating compound obtained by methanol extraction of erythrina abicinica and purified by pure separation using chromatography and a compound obtained by efficiently extracting and purifying it as an active ingredient ( It relates to a composition for the prevention and treatment of metabolic syndrome.

Recently, due to the improvement of living standards according to economic development, the hygiene environment is improved and the dietary life is continuously improved. However, the amount of calories consumed due to excessive food, while the amount of calories consumed due to lack of exercise is showing a tendency to increase obesity. Obesity not only harms young people's skin-like beauty, but also keeps obesity alive, leading to various diseases such as hypertension, diabetes, hyperlipidemia and coronary artery disease, as well as breast cancer, uterine cancer and colon cancer. It is reported to cause it and is now treated as a fatal disease [J. Biol. Chem., 273, 32487-32490 (1998); Nature, 404, 652-660 (2000).

Current treatments for treating obesity can be divided into drugs that affect the central nervous system and affect appetite and drugs that inhibit absorption by acting on the gastrointestinal tract. Drugs that act on the central nervous system include drugs such as fenfluramine and dexfenfluramine that inhibit the serotonin (5-HT) nervous system according to each mechanism, drugs such as ephedrine and caffeine through the noradrenaline nervous system, serotonin and noradrenaline nervous system Drugs such as sibutramine that act and inhibit obesity are commercially available. In addition, orlistat, which inhibits intestinal lipase by acting on gastric / intestinal tract and inhibits obesity, is used as a typical drug. However, drugs such as fenfluramine, which have been used previously, have been banned due to side effects such as primary pulmonary hypertension and heart valve lesions, and other drugs have also caused problems such as decreased blood pressure and lactic acidosis, which can lead to heart failure and kidney disease. The patient has a problem that cannot be used.

  The present inventors have therefore become interested in the mechanisms of regulating energy metabolism in recent years to find more improved methods for treating obesity. Usually, metabolic syndrome prevention and treatment should be taken for a long time after the onset of the disease, and the energy-regulatory organs are mitochondrial, and the energy regulator for this should be higher safety (low toxicity) than other drug targets. I came to explore.

AMPK is a phosphatase that regulates activity in response to changes in the energy state (ATP / AMP ratio) that is changed by the nutritional state, exercise, or stress of the cell. Glucose transport, fatty acid synthesis, cholesterol biosynthesis ( cholesterol synthesis) and various physiological effects are known [Ann. Rev. Pharmacol. Toxicol., 47, 185-210 (2007).

Recent reports have shown that AMPK is involved in glucose uptake of muscle cells stimulated by exercise, and thus AMPK generally plays a role as a sensor to monitor energy levels in cells. In exercise or hunger, muscle cells, hepatocytes and adipocytes inhibit the synthesis of fat and glycogen to provide the necessary energy, and generate the energy needed by the body through lipolysis from storage materials (fat). AMPK is involved in doing this. AMPK is also known as an intracellular signaling material of leptin and adiponectin secreted from adipocytes. In particular, adiponectin is measured at low concentrations in the blood of obese humans compared to normal weight humans, suggesting that AMPK is highly associated with insulin resistance due to obesity, and therefore AMPK activator is an excellent drug for obesity. Emerging as a target point [Nat. Rev Mol Cell Biol., 8 (10), 774-785 (2007)].

Many companies have been developing drugs targeting these drugs since it has been found that the drug targets of metformin, which have been used to treat oral diabetes but do not know the mechanism, are related to AMPK activity. In 2008, a team published in Australia's Garvan Institute for Medical Research reported that four components derived from bitter melon regulate metabolism in the body by activating AMPK [Chem Biol , 15 (5), 263-273 (2007).

In general, among the various methods for the development of drugs with new ingredients, finding new active ingredients from natural medicines used in traditional medicine is highly likely to succeed in drug development, rather than the development of drugs by experimental modification of existing drugs. In addition, since these active ingredients have been used for a long time, there is little concern about toxicity due to drugs, and thus there is an advantage that they can be used as a drug treatment.

Erythrina abyssinica , which is the subject of the present invention, belongs to the legume and the tree of the genus Erysrina is a well-known plant of about 110 species. The genus Erislina grows across tropical and subtropical regions, and the subject of the present invention is Erislina abyssinica, which is a tree native to Cameroon in central Africa and has red flowers. The plant extract of Erysri abisinica has been used as a medicinal plant in Africa since ancient times, and is known to be particularly effective against antibacterial diseases. The present inventors confirmed the structure by separating seven kinds of compounds having an action of activating the AMPK enzyme from erythrina abysinica. These compounds were coumestan-based compounds, pterocarpan-based compounds and benzofuran-based compounds. Since these compounds commonly activated the AMPK enzyme, it was found that the compounds of the present invention can be used for the prevention and treatment of metabolic syndrome including obesity.

An object of the present invention is to provide compounds that have an AMPK activating action derived from erythrina abicinica and exhibit prophylactic and therapeutic effects on obesity or metabolic syndrome-related diseases.

It is another object of the present invention to provide a composition containing as an active ingredient a mixture of two or more compounds having the prophylactic and therapeutic effect of obesity or metabolic syndrome having an AMPK activating action derived from erythri or abysinica.

In view of the above, the present inventors collected various native plants and herbal medicines to investigate the AMPK activation activity, and finally selected erythrina or abyssinica as candidate plants. Thereafter, the plant is extracted with an organic solvent to act as an AMPK activating compound represented by the following formula (1), erythribysin F (erythribyssin F, compound 1), erythribysin N (erythribyssin N, compound 2), erythribinine Erythribyssin O (compound 3), sigmoidin K (compound 4), isosojagol (compound 5), eryvarin D (compound 6), erypoegin F , Compound 7) was extracted.

The compound is characterized in that it is used for at least one selected from a prophylactic or therapeutic substance associated with obesity or metabolic syndrome and a pharmaceutical substance for AMPK activation.

The compound may be derived from Erysri or Abyssinica.

In addition, the compound may be derived from the methanol extract of Erysri or Abyssinica. The methanol extract can then be further fractionated with ethyl acetate.

The composition for the prevention or treatment of obesity or metabolic syndrome related diseases according to the present invention is characterized in that it comprises at least one compound of the above compounds as an active ingredient.

The compound is,

Extracting and fractionating erythrina abicinica with an organic solvent;

Purely separating and purifying the compound that activates AMPK using the active compound chromatography;

Examining the chemical structure and physicochemical properties of the compound obtained in the step; And

Differentiating a C2C12 cell line, which is a muscle progenitor, and examining AMPK activation of the compound of the above step;

Can be obtained through the process of.

The structural confirmation of the compounds can be confirmed from the interpretation of physicochemical properties and nuclear magnetic resonance spectra.

The AMPK activating compound according to the present invention is extracted from erythrina or abysinica by an organic solvent (alcohol, ether, acetone, etc.), partitioning ethyl acetate and water, using an adsorption resin such as diion HP-20 resin, column chromatography, and the like. A well-known method used for the separate extraction of plant components, such as a method by the above, can be easily obtained by combining individually or suitably. The crude extract can be further purified according to a commercial method as needed.

The chromatography used in the present invention includes silica gel column chromatography, L-20 column chromatography, thin layer chromatography (TLC) and high performance liquid chromatography (TLC). high performance liquid chromatography) and the like.

Since the compound according to the present invention acts on AMPK activation, it is effective in preventing or treating obesity or metabolic syndrome and can be advantageously utilized due to its high bioavailability.

The compound according to the present invention can be easily separated from erythrica or abyssin and also has high stability, and thus can be used as an additive for food and medicine.

Pharmaceutical compositions comprising extracts according to the invention, in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterile injectable solutions, respectively, according to conventional methods. Can be formulated and used. Examples of carriers, excipients and diluents that can be included in the composition containing the extract include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. 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 preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient in the extract, for example, starch, calcium carbonate, sucrose or lactose, gelatin, and the like. Are mixed to prepare. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The dosage of the active ingredient will vary depending on the age, sex, weight of the subject to be treated, the particular disease or pathology to be treated, the severity of the disease or pathology, the route of administration and the judgment of the prescriber. Dosage determination based on these factors is within the level of skill in the art and generally dosages range from 0.01 mg / kg / day to approximately 2000 mg / kg / day. More preferred dosage is 1 mg / kg / day to 500 mg / kg / day. Administration may be once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect. The extract of the present invention can be administered to mammals such as mice, livestock, humans, etc. by various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection. Since the extract of the present invention has little toxicity and side effects, it is a drug that can be used safely even when taken for long periods of time.

In addition, the present invention provides a health functional food for the prevention of obesity, including the erythrina abicinica extract and a food supplement acceptable food supplement. The health functional food of the present invention includes the form of tablets, capsules, pills or liquids, and the food to which the compound of the present invention can be added, for example, various foods, beverages, gums, teas, vitamin complexes, etc. And health functional foods. In detail, the present invention provides a dietary supplement for the prevention and treatment of obesity or metabolic syndrome, including an extract containing erythrina abicinica extract as an active ingredient and a food supplement acceptable food supplement.

Compositions comprising compounds 1 to 7 according to the present invention can be expected to be effective in the prevention and treatment of obesity or metabolic syndrome through the action on energy metabolism by activating the AMPK enzyme in the cell.

In addition, since it is extracted and purified from the natural product erythrii or abysinica, which has been used as a food raw material such as herbal medicines and edible tea, it has a low cytotoxicity and can be used and applied to medicines, cosmetics, and food industries.

FIG. 1 shows the ¹³ C-NMR spectrum of a novel compound erythricisin N (Compound 1) activating the AMP-activated protein kinase enzyme (AMPK) obtained from erythrina abysinica.
FIG. 2 shows ¹³ C-NMR spectra of the novel compound erythrivisin F (Compound 2) activating the AMP-activated protein kinase enzyme obtained from erythrina abysinica.
Figure 3 shows the ¹³ C-NMR spectrum of the novel compound erythricisin O (Compound 3) activating the AMP-activated protein kinase enzyme obtained from erythrina abysinica.
Figure 4 shows the results of Western blot analysis of the activity of AMPK enzymes through the expression of p-AMPK and p-ACCs by treating each of the active compounds obtained from Erysri or Abysinica in differentiated C2C12 cell lines.

Hereinafter, a preferred embodiment of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the concept of the invention to those skilled in the art.

Example 1 Isolation and Purification of the Active Compound from Erysri or Abyssinica

5 kg of the shell of erysri abisinica was extracted three times each day at room temperature using 10 liters of methanol. The methanol extract was concentrated under reduced pressure, and then 350 g of the extract was suspended in water, and then solvent fractionation was performed sequentially with n-hexane, ethyl acetate, and butanol, and AMPK activity was measured for each solvent fraction. Indicated. The ethyl acetate fractions were concentrated under reduced pressure, and 156 g of the fractions were confirmed. The ethyl acetate fraction (45 g) was subjected to silica gel column chromatography under a solvent gradient of hexane-acetone (10: 1 to 0: 1) to obtain five fractions (fr.1-5). Fraction F2 (2.4 g) was collected in 200 ml portions under a solvent gradient of methanol-water (6: 4 → 1: 0) to obtain 5 small fractions (fr.2.1-2.5). Small fraction F.2.3 was prepared using HPLC [Optima Pak C ₁₈ column 20 × 150 mm over 30 minutes using 70% acetonitrile (MeCN, acetomitrile); 10 μm particle size 2 ml / min; UV detection: 254 nm] was purely separated from compound 1 (4.7 mg; t _R = 24.5 min) and compound 7 (27.8 mg; t _R = 26.8 min). Small fraction F.2.4 was prepared by HPLC [Optima Pak C _{18 using} 78% acetonitrile for 25 minutes in solvent. column 20 × 150 mm; 10 μm particle size 2 ml / min; UV detection: 254nm] was carried out in the separation of the compound _{6 (227.8mg, t R = 19.0min} ) and compound _{3 (5.8mg, t R = 22.5min} ). Fraction F3 (4 g) was collected in 300 ml portions under a solvent gradient of methanol-water (5: 5 → 1: 0) to obtain five small fractions (fr.3.1-3.5). Subfraction F.3.3 was prepared using HPLC [Optima Pak C ₁₈ column 20 × 150 mm in 60% acetonitrile; 10 μm particle size 2 ml / min; UV detection: 254 nm] was carried out to obtain compound 5 (3.7 mg, t _R = 14.5 min) and compound 2 (10.8 mg; t _R = 20.8 min). In small fraction F.3.3, Compound 4 (15 mg) was isolated purely using silica gel column chromatography under a solvent condition of hexane-acetone (4: 1).

Table 1 shows the results of measuring the AMPK activation of the erythrina abicinica extract solvent fractions. The degree of AMPK activation was the strongest in the ethyl acetate fraction.

Erysrina Abysinica Solvent Fraction
AMPK activation concentration (µg / ml)
Methanol Extract (A Fraction)
200
n-hexane (B fraction)
500
Ethyl acetate (C fraction)
50
Butanol (D fraction)
300

< Example  2: Physical and chemical properties and chemical structure analysis of compounds>

It was confirmed from the ¹ H, ¹³ C-NMR analysis that the compound isolated from the shell of the erythrina abysinica had two pairs of aromatic hydrogens and compounds 1 to 3 were identified as novel compounds.

Compound 1 was obtained as a white powder and the molecular formula was determined as C ₂₁ H ₁₈ O ₅ from a measurement of 350.1154 in a high mass spectrometer (HRESIMS). Hydrogen nuclear magnetic resonance spectra ( ¹ H NMR) indicate δ _H 3.68 (2H, d), 5.36 (1H, m), 1.67 (3H, s), 1.91 (3H, s) and ABX- suggesting the presence of prenyl groups The directional protons in the form of δ _H 6.96 (1H, br, s), 7.02 (1H, br, d) and 7.91 (1H, d) and a pair of ortho linked δ _H 7.17 (1H, d, J) = 8.4 Hz) and 7.74 (1H, d, J = 8.4 Hz). _C-9 (δ C 157.3) and _C-9 (δ C 157.3) from the methylene protons of δ _H 3.68 were identified to be from the connection from the methoxy protons is measured at δ _H 3.96 the position of each substituent is etched embisi (HMBC), From the observations of C-10 ( δ _C 115.1) and C-10a ( δ _C 156.5), the compound was 3-hydroxy-9-methoxy-10- (3-methylbut-2-enyl) coumestan. (erythribyssin N, compound 1).

Compound 2 was obtained as a brown powder and the UV spectrum showed absorption bands at 329nm and 345nm, which are characteristic of 2-arylbenzofuran. From 445.1626, a mass spectrometer measurement, the molecular formula was identified as C ₂₅ H ₂₆ O _{6. From the} carbon nuclear magnetic resonance spectrum ( ¹³ C NMR), the olefinic quaternary carbon characteristic of 2-arylbenzofuran was identified by δ _C 159.1 and δ. _It is identified in _C 111.1. Rise from the hydrogen nuclear magnetic resonance spectrum bovine aromatic hydrogen _{δ H 7.70 (1H, d,} J = 7.5, 4-H) and 6.92 (1H, d, J = 7.5, 5-H), 2 a single aromatic hydrogen δ _H Two prenyl groups were also identified at 6.56 (1H, s, 3′-H) and 7.34 (1H, s, 6′-H). The final position of each substituent is an HMBC resulting in one prenyl group linked from H-1 ″ to C-6, C7, C-7 and C-8 from the link to the other prenyl group H-1 ″. From C 'it was found to connect from C-1' to 5 'from C-4', C-5 'and H-6'. Compound 2 is 2- [2 ', 4'-dihydroxy-5'-(3-methylbut-2-enyl) phenyl] -6-hydroxy-7- (3-methylbut-2-enyl) -1-benzofuran-3 -Oic acid was named erythribyssin F (Compound 2).

Compound 3 was obtained as a yellow powder and its molecular weight was determined to be C ₂₁ H ₁₈ O ₅ from the results of a high mass spectrometer measured at 350.1154. From the hydrogen nuclear magnetic resonance spectrum, δ _H 5.75 (2H, s), which is a pterocarpene hydrogen at position 6, was confirmed. In addition, hydrogen and prenyl groups in an aromatic ortho relationship having aromatic monohydrogen having J = 8.4 Hz at δ _H 6.89 and 7.18 were identified from δ _H 6.45 and 7.82. Aldehyde groups present in novel compound 3 were identified in δ _C 196.1 by δ _H 9.94 (1H, s) from the hydrogen nuclear magnetic resonance spectrum and corresponding carbon measurements from the carbon nuclear magnetic resonance spectrum. Finally, the structure was confirmed by locating aldehyde ( δ _H 9.94), C-2 ( δ _C 116.6), and C-3 ( δ _C 164.9). Thus, compound 3 was named erythribyssin O (compound 3) as 3,9-dihydroxy-10-prenyl-2-formyl-6a, 11a-dehydroxypterocarpan.

1 to 3 show ¹³ C NMR spectra of Compounds 1 to 3. FIG.

Compounds 4 to 7, which have been previously reported but whose AMPK activating activity was first identified, are sigmoidin K (compound 4), isosojagol (compound 5), eryvarin D (compound 6). And erypoegin F (compound 7).

Compounds 4 to 7 were confirmed to have structural and physicochemical properties by comparing hydrogen nuclear magnetic resonance spectra, carbon nuclear magnetic resonance spectra with mass spectrometry and UV absorption spectra.

2-1. Erythribyssin N (compound 1)

Appearance: white powder

Molecular Weight: 350.1154

Molecular Formula: C ₂₁ H ₁₈ O ₅

UV absorption spectrum ( c 0.02, MeOH): λ _max nm: 210, 214, 242, 299, 336, 352 nm

Hydrogen nuclear magnetic resonance spectrum (500 MHz, acetone- d ₆ ): δ 7.91 (1H, d, J = 9.0, H-1), 7.74 (1H, d, J = 8.4, H-7), 7.17 (1H, d, J = 8.4, H-8), 7.02 (1H, br d, J = 7.8, H-2), 6.96 (1H, br s, H-4), 5.36 (1H, t-like, J = 7.2 , H-2 '), 3.96 (3H, 9-OCH ₃ ), 3.68 (2H, d, J = 7.2, H-1'), 1.91 (3H, s, H-5 '), 1.67 (3H, s , H-4 ′);

Carbon nuclear magnetic resonance spectrum (125 MHz, acetone- d ₆ ): δ 161.4 (C-11a), 162.9 (C-3), 158.6 (C-6), 157.3 (C-9), 156.5 (C-10a) , 155.5 (C-5), 132.9 (C-3 '), 123.7 (C-1), 122.4 (C-2'), 119.0 (C-7), 117.8 (C-6b), 115.1 (C-10 ), 114.7 (C-8), 110.2 (C-2), 105.7 (C-11b), 104.3 (C-4), 103.5 (C-6a), 25.9 (C-4 '), 23.4 (C-1 ′), 18.0 (C-5 ′).

2-2. Erythribyssin F (compound 2)

Appearance: Brown Powder

Molecular Weight: 445.1626

Molecular Formula: C ₂₅ H ₂₆ O ₆

UV absorption spectrum ( c 0.025, MeOH): λ _max nm: 329, 345 nm

Hydrogen nuclear magnetic resonance spectrum (500 MHz, acetone- d ₆ ): 7.70 (1H, d, J = 7.5, H-4), 7.34 (1H, s, H-5 '), 6.92 (1H, d, J = 7.5, H-5), 6.56 (1H, s, H-2 ′), 5.39 (1H, m, H-2 ″), 5.36 (1H, m, H-2 ″ ′), 3.59 (2H, d, J = 7.0, H-1 ″), 3.31 (2H, d, J = 7.0, H-1 ″ ′), 1.83 (3H, s, H-5 ″), 1.72 (6H, s, H-4 ″ ′ And H-5 &quot;'), 1.66 (3H, s, H-4 &quot;);

Carbon nuclear magnetic resonance spectrum (125 MHz, acetone- d ₆ ): δ 168.9 (-COOH), 159.1 (C-2), 158.9 (C-4 '), 156.5 (C-2'), 154.6 (C-8 ), 153.6 (C-6), 132.8 (C-6 '), 132.5 (C-3 ″'), 132.2 (C-3 ″), 124.0 (C-2 ″ ′), 123.1 (C-2 ″) , 121.1 (C-5 '), 120.7 (C-9), 120.4 (C-4), 113.6 (C-5), 119.9 (C-7), 112.0 (C-1'), 111.1 (C-3 ), 105.5 (C-3 ′), 28.4 (C-1 ″ ′), 26.0 (C-4 ″ ′), 25.9 (C-4 ″), 23.3 (C-1 ″), 18.1 (C-5 ″ ), 17.9 (C-5 ″ ′).

2-3. Erythribyssin O (compound 3)

Appearance: Yellow Powder

Molecular Weight: 350.1154

UV absorption spectrum ( c 0.025, MeOH): λ _max nm: 210, 214, 242, 299, 336, 352 nm

Hydrogen nuclear magnetic resonance spectrum (500 MHz, acetone- d ₆ ): δ 9.94 (1H, -CHO), 7.82 (1H, s, H-1), 7.18 (1H, d, J = 8.4, H-7), 6.89 (1H, d, J = 8.4, H-8), 6.45 (1H, s, H-4), 5.75 (2H, s, H-6), 5.40 (1H, m, H-2 ′), 3.64 (2H, d, J = 7.2, H-1 '), 1.88 (3H, s, H-5'), 1.67 (3H, s, H-4 ');

Carbon nuclear magnetic resonance spectrum (125 MHz, acetone- d ₆ ): δ 196.1 (2-CHO), 162.9 (C-3), 161.4 (C-11a), 158.6 (C-6), 157.3 (C-9) , 156.5 (C-10a), 155.5 (C-4a), 132.9 (C-3 '), 123.7 (C-1), 122.4 (C-2'), 119.0 (C-7), 117.8 (C-6b ), 115.1 (C-10), 114.7 (C-8), 110.2 (C-2), 105.7 (C-11b), 104.3 (C-4), 103.5 (C-6a), 25.9 (C-5 ' ), 23.4 (C-1 ′), 18.0 (C-4 ′).

2-4. Sigmoidin K (compound 4)

Appearance: Brown

UV absorption spectrum ( c 0.025, MeOH): λ _max nm: 210, 242, 288, 316, 352 nm

Hydrogen Nuclear Magnetic Resonance Spectrum (500MHz, acetone- d ₆ ): δ 7.72 (1H, s, H-1), 7.59 (1H, d, J = 8.0, H-7), 7.04 (1H, d, J = 8.0 , H-8), 6.96 (1H, s, H-4), 5.44 (1H, t-like, J = 7.5, H-2 ''), 5.40 (1H, t-like, J = 7.5, H-2 ′), 3.69 (2H, d, J = 7.5, H-1 ′), 3.44 (2H, d, J = 7.5, H-1 ″), 1.95 (3H, s, H-5 ′), 1.81 (3H , s, H-5 ″), 1.77 (3H, s, H-4 ″), 1.69 (3H, s, H-4 ′);

Carbon nuclear magnetic resonance spectrum (125 MHz, acetone- d ₆ ): δ 160.8 (C-11a), 159.5 (C-3), 158.7 (C-6), 156.1 (C-9), 154.9 (C-10a) , 154.5 (C-4a), 134.1 (C-3 ″), 132.6 (C-3 ′), 127.4 (C-2), 122.6 (C-2 ″ and C-2 ′), 122.2 (C-1) , 118.9 (C-7), 116.8 (C-6b), 114.4 (C-8), 113.4 (C-10), 105.9 (C-11b), 103.9 (C-6a), 28.4 (C-1 ″) , 26.1 (C-5 ″), 26.0 (C-4 ′), 23.5 (C-1 ′), 18.0 (C-5 ′), 17.9 (C-4 ″).

2-5. Isosojagol (compound 5)

Appearance: Brown Powder

UV absorption spectrum ( c 0.01, MeOH): λ _max nm: 208, 242, 316, 343 nm

Hydrogen nuclear magnetic resonance spectrum (500 MHz, acetone- d ₆ ): δ 7.92 (1H, d, J = 9.0, H-1), 7.02 (1H, dd, J = 9.0, 2.5, H-2), 6.94 ( 1H, d, J = 2.5, H-4), 7.60 (1H, d, J = 8.5, H-7), 7.05 (1H, d, J = 8.5, H-8), 5.42 (1H, m, H -2 '), 3.69 (2H, d, J = 7.0, H-1'), 1.90 (3H, s, H-5 '), 1.67 (3H, s, H-4');

Carbon Nuclear Magnetic Resonance Spectrum (125 MHz, acetone- d ₆ ): δ 162.5 (C-3), 161.1 (C-11a), 158.9 (C-6), 156.6 (C-9), 155.9 (C-10a) , 154.9 (C-4a), 132.3 (C-3 '), 123.2 (C-2'), 122.9 (C-1), 119.2 (C-7), 117.1 (C-6b), 114.7 (C-10 ), 114.5 (C-8), 111.1 (C-2), 105.5 (C-11b), 104.0 (C-4), 103.7 (C-6a), 26.1 (C-4 '), 23.7 (C-1 ′), 18.0 (C-5 ′).

2-6. Eryvarin D, compound 6

Appearance: Brown Powder

Hydrogen Nuclear Magnetic Resonance Spectrum (500 MHz, acetone- d ₆ ): δ 8.70 (1H, br s, 3-OH), 7.34 (1H, d, J = 8.0, H-1), 7.24 (1H, d, J = 8.5, H-7), 6.95 (1H, d, J = 8.5, H-8), 6.53 (1H, d, J = 8.0, H-2), 6.44 (1H, d, J = 2.5, H- 4), 5.54 (1H, s, H-6), 5.33 (2H, m, H-2 ′), 3.89 (3H, 9-OCH ₃ ), 3.60 (2H, d, J = 7.5, H-1 ′ ), 1.87 (3H, s, H-5 '), 1.66 (3H, s, H-4');

Carbon Nuclear Magnetic Resonance Spectrum (125 MHz, acetone- d ₆ ): δ 159.9 (C-3), 156.4 (C-4a), 155.9 (C-9), 155.4 (C-10a), 148.3 (C-11a) , 132.3 (C-3 '), 123.0 (C-2'), 122.0 (C-1), 120.5 (C-6b), 116.8 (C-7), 114.6 (C-10), 109.7 (C-11b ), 109.5 (C-2), 108.9 (C-8), 106.7 (C-6a), 104.6 (C-4), 66.1 (C-6), 57.0 (9-OCH ₃ ), 25.9 (C-4 ′), 23.5 (C-1 ′), 18.0 (C-5 ′)

2-7. Erypoegin F (compound 7)

Appearance: Brown Powder

UV absorbance spectrum ( c 0.02, MeOH): λ _max nm: 219, 289, 331, 346 nm

Hydrogen nuclear magnetic resonance spectrum (500 MHz, acetone- d ₆ ): 10.17 (1H, s, -CHO), 7.91 (1H, d, J = 8.5, H-4), 7.53 (1H, d, J = 8.5, H-6 ′), 7.07 (1H, d, J = 8.5, H-5), 6.61 (1H, dd, J = 8.5, 2.0, H-5 ′), 6.65 (1H, d, J = 2.0, H -2 '), 5.34 (1H, t-like, J = 7.5, H-2 "), 3.59 (2H, d, J = 7.5, H-1"), 1.81 (3H, s, H-5 ") , 1.64 (3H, s, H-4 ″);

Carbon nuclear magnetic resonance spectrum (125 MHz, acetone- d ₆ ): δ 188.1 (-CHO), 164.0 (C-2), 162.1 (C-4 '), 157.9 (C-2'), 156.5 (C-6 ), 154.5 (C-8), 133.5 (C-6 ′), 132.5 (C-3 ″), 123.5 (C-2 ″), 120.1 (C-4), 119.9 (C-9), 117.8 (C -3), 114.1 (C-7), 109.9 (C-5), 109.1 (C-5 '), 109.0 (C-1'), 104.2 (C-3 '), 25.9 (C-4 "), 23.4 (C-1 ″), 18.0 (C-5 ″).

< Example  3: Erisrina From Abyssinica  Of isolated compounds 1 to 7 AMPK  Measurement of Activation Action>

Compound 1 to Compound 7 purified from erythrina abysinica In order to measure the efficacy of AMPK activation, Western blot was performed using a C2C12 myoblast cell line. C2C12 cells were seeded in 6-wells plates and then cultured using DMEM medium containing 10% bovine serum and then replaced with DMEM medium containing 1% FBS to induce differentiation. After confirming the differentiation of the cells and maintained in DMEM medium without serum for 16 hours, each compound was treated at a final concentration of 5μM and treated with 100μM of Aicar as a positive control, and then incubated for 2 hours Were collected and disrupted by ultrasonication to obtain a protein. Aicar (aminoimidazole carboxamide ribonucleotide) was used as a positive control for AMPK activity, and DMSO (within 0.1% of cell medium) as a solvent of the compound was used as a negative control. Thereafter, 10% SDS-PAGE electrophoresis was performed and the protein was transferred to the PVDF membrane. After maintaining at room temperature for 1 hour with 5% skim milk, it was maintained at 4 ℃ for 18 hours or more using a phosphorylated AMPK (phospho-AMPK, Thr172) antibody and using this to determine the amount of protein expression. Increased AMPK activity during energy metabolism is known to increase phosphorylation of ACCs (acetyl-CoA carboxylase 1 and 2), so phosphorylated ACCs (phospho-ACCs) using proteins obtained by the same method to confirm AMPK activation. It was confirmed.

The results of measuring the AMPK activating action of Compounds 1 to 7 isolated from erythrina abysinica are shown in FIG. 4.

Referring to FIG. 4, Compound 1, Compound 4, and Compound 5, which are comumestan-based compounds, showed the strongest activity, and other compounds also showed strong AMPK activity.

< Example  4 : DIO ( Diet - Induced Obesity ) Model Anti-obesity  Effect Black>

[Step 1] Sample Preparation

Compound 2 and compound 4 used in animal experiments were orally administered daily at a concentration of 100 mg / kg to confirm the anti-obesity effect, and the drug administered before oral administration was prepared by dissolving in 5% aqueous solution of methyl cellulose (Sigmaco.USA).

[Step 2] Experiment group setting and weight loss effect verification

In order to conduct the experiment, 10 rats were prepared per group of 7-week-old male C57BL / 6J mice, and after 1 week of adaptation, they were separated into individual cages and divided into 12-hour daytime (7 am-5pm). ) And nightly cycles. The experimental group was divided into three groups: 1) a high-fat diet group, 2) a high-fat diet and a compound 2 100 mg / kg / day intake group, and 3) a high-fat diet and a compound 4 100 mg / kg / day intake group. Each dose was administered orally at a constant time (10 AM) for 8 weeks. Body weight was measured once a week (11 AM), and analyzed by measuring the final body weight of the experimental group and the control group 8 weeks after the start of administration. Table 2 shows the results of measuring weight change after oral administration of each substance.

group

Weight change after oral administration (g) 14 days 21st 28 days 35 days 42 days 49 days 56 days
High-fat diet 4.45
± 0.45 6.23
± 0.23 8.56
± 0.78 10.45
± 0.34 11.56
± 0.86 12.32
± 0.23 13.78
± 0.76 Compound 2+
High-fat diet 2.75
± 0.56 3.43
± 0.75 4.19
± 0.34 6.31
± 0.34 7.24
± 0.34 8.21
± 0.23 8.45
± 0.45 Compound 4+
High-fat diet 2.62
± 0.23 3.33
± 0.34 4.45
± 0.45 6.43
± 0.75 7.64
± 0.74 8.34
± 0.64 8.23
± 0.63

Comparing the body weight of each administration group at 56 days after the experiment, the weight gain of the group ingesting Compound 2 or Compound 4 together with the high-fat diet was higher than that of the high-fat diet as shown in Table 2. Noticeably low. The body weight of the high-fat diet group increased by 13.78 g, while the weight of the compound 2 group increased by 8.45 g and the body weight of the compound 4 group increased by 8.23 g. These weight gains were statistically significant at the 95% confidence level compared to the high-fat diet group.

Example 5 Toxicity Test

5-1. Acute Toxicity

To investigate the toxicity to animals in acute (within 24 hours) of excessively ingesting the active fraction extracted from Erysri abisinica and the fraction containing Compound 1 to Compound 7, and to determine the mortality rate The experiment was performed. 40 ICR mouse strains, which are common mice, were assigned to 10 control groups and 10 experimental groups. Only 30% PEG-400 was administered to the control group, and the experimental group orally administered the active fraction extracted from Erysri abicinica and the fraction containing Compound 1 to Compound 7 at a concentration of 1.0 g / kg, respectively. After 24 hours of administration, the mortality was examined, and both the control group, the active fraction extracted from 1.0g / kg erythrina abisinica, and the experimental group containing the compound 1 to compound 7 survived.

5-2. Long-term and tissue toxicity experiments in experimental and control groups

In the long-term toxicity experiment, C57BL / 6J mice were treated with an active fraction extracted from erythrina abisinica and a fraction containing compound 1 to compound 7 to investigate the effect on each organ (tissue) of the animal. Blood was collected after 8 weeks from animals in the control group administered with the bay, and blood concentrations of glutamate-pyruvate transferase (GPT) and blood urea nitrogen (BUN) were measured using a Select E (Vital Scientific NV, Netherland) instrument. As a result, in the case of GPT, which is known to be related to hepatotoxicity, and BUN, which is known to be related to nephrotoxicity, there was no difference in the experimental group compared to the control group. In addition, liver and kidneys were removed from each animal, and histological observations were performed by optical microscopy through a conventional tissue fabrication process. No abnormalities were observed.

Example 6 Pharmaceutical Formulation Example

6-1. Manufacture of tablets

200 g of the erythrina abicinica extract according to the present invention or a compound isolated therefrom was mixed with 175.9 g of lactose, 180 g of potato starch, and 32 g of colloidal silicic acid. 10% gelatin solution was added to the mixture, which was then ground and passed through a 14 mesh sieve. It was dried and the mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into a tablet.

6-2. Preparation of Injection

1 g, 0.6 g of sodium chloride, and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml of the extract of erythrina abicinica or a compound isolated therefrom according to the present invention. The solution was bottled and sterilized by heating at 20 ° C. for 30 minutes.

Example 7 Food Preparation Example

7-1. Preparation of Cooking Seasonings

Erisrina abicinica extract according to the present invention or a compound isolated therefrom was added 0.2 to 10% by weight, respectively, to prepare a cooking seasoning for health promotion.

7-2. Manufacture of flour food products

0.1 to 5.0% by weight of each of the erythri abicinican extract or the compound isolated therefrom is added to the flour, and bread, cake, cookies, crackers and noodles are prepared using the mixture to prepare foods for health promotion. Prepared.

7-3. Preparation of soups and gravy

0.1-1.0 wt% of erythrina abiciniica extract or a compound isolated therefrom according to the present invention was added to soups and broths to prepare meat products for health promotion, soups and broths of noodles.

7-4. Manufacture of dairy products

0.1-1.0% by weight of each of the erythri abicinicar extract or the compound isolated therefrom according to the present invention was added to milk, and various dairy products such as butter and ice cream were prepared using the milk.

Example 8 Beverage Preparation Example

8-1. Vegetable Juice Manufacturing

Health promotion vegetable juice was prepared by adding 0.5 g of the erythrina abicinica extract according to the present invention or the compound isolated therefrom to 1,000 ml of tomato or carrot juice.

8-2. Fruit juice manufacturing

Health promotion fruit juice was prepared by adding 0.1 g of erythrina abicinica extract or a compound isolated therefrom to 1,000 ml of apple or grape juice.

Claims (9)

Prevention of obesity or metabolic syndrome characterized in that it contains an extract of Erythrina abyssinica containing at least one of sigmoidin K (compound 4) and isosogol (compound 5) as an active ingredient Therapeutic pharmaceutical composition.

delete delete delete For improvement of obesity or metabolic syndrome, characterized by containing Erythrina abyssinica extract containing at least one of sigmoidin K (compound 4) and isosogol (compound 5) as an active ingredient Health functional food.

delete The method of claim 1,
The pharmaceutical composition is a pharmaceutical composition for the prevention or treatment of obesity or metabolic syndrome, characterized in that to increase the activity of AMP-activated protein kinase (AMPK) enzyme. A pharmaceutical composition for preventing or treating obesity or metabolic syndrome, comprising at least one of sigmoidin K (compound 4) and isosogol (compound 5) as an active ingredient.

The method of claim 8,
The composition is a pharmaceutical composition for the prevention or treatment of obesity or metabolic syndrome, characterized in that to increase the activity of AMP-active protein kinase (AMPK) enzyme.

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