KR20140045647A - Compositions for the prevention or treatment of metabolic diseases or complications thereof for containing pheophorbide compounds or pharmaceutically acceptable salts thereof as an active ingredient - Google Patents

Abstract

The present invention relates to a composition comprising a pheophorbide compound, a tautomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient for preventing or treating metabolic diseases or complications thereof. More specifically, the pheophorbide compound according to the present invention effectively inhibits an activity of alpha-glucosidase and has little or no toxicity, thereby enabling the compound to be used for the prevention or treatment of the metabolic diseases including diabetes or complications thereof. [Reference numerals] (AA) Structure of compound 1

Description

Compositions for the prevention or treatment of metabolic diseases or complications thereof containing the fairovide-based compound or a pharmaceutically acceptable salt thereof as an active ingredient {including compositions for the prevention or treatment of metabolic diseases or complications} for containing Pheophorbide compounds or acceptable salts pretty as an active ingredient}

The present invention relates to a composition for the prophylaxis and treatment of metabolic diseases or complications thereof comprising a phenophorbide compound, a tautomer thereof or a pharmaceutically acceptable salt thereof as an active ingredient.

Due to the rapid development of modern society and the abundance of nutritional status, enormous environmental changes have occurred in the metabolic organ of the human body, which is not different from primitive age. The prevalence of metabolic diseases in which two or more diseases such as obesity, type 2 diabetes, hypertension, hypertriglyceridemia, hypercholesterolemia, and arteriosclerosis are combined is called the geriatric disease or modern disease according to the 2005 National Health and Nutrition Survey (32.9% of males and 31.8% of females), and the heart disease and stroke that are caused by it are increasing to occupy the second or third place of Korean deaths.

It is known as insulin resistance syndrome, which is caused by loss of metabolic waste and toxins caused by inadequate balance of metabolism and waste of human body due to loss of toxins. It develops into Metabolic Syndrome. Metabolic Syndrome gives damage to the coronary artery and provides the cause of heart disease or paralysis. It is not capable of removing salt from the kidneys, causing hypertension, providing the cause of cardiovascular disease It is known that it increases the triglyceride ratio, increases the risk of blood clotting, and also decreases insulin production by type 2 diabetes, causing damage to eyes, kidneys, and nerves.

Diabetes mellitus is a disease in which glucose is released into the urine due to its higher blood sugar concentration than normal people. It is caused by abnormal metabolic processes or abnormal physiological activity of insulin in β-cells of Langerhans islets in the pancreas. Diabetes is divided into insulin-dependent diabetes mellitus and non-insulin dependent diabetes mellitus, depending on the secretion and action of insulin. Insulin-dependent diabetes mellitus (type 1 diabetes) is a severe insulin deficiency because the pancreatic beta cells are destroyed and cannot secrete insulin. Therefore, insulin-dependent diabetes mellitus should be supplied externally regularly to prevent ketoneosis and death. It occurs in so called pediatric diabetes. On the other hand, insulin-independent diabetes (type 2 diabetes), which is more than 90% of all diabetic patients, is capable of secreting insulin from the beta cells of the pancreas but is a disease caused by a dysfunctional state of insulin. Diabetes is a hyperglycemic environment in the body that causes high osmotic stress, leading to complications such as cataracts and kidney disease (Campbell, RK and Steil, CF 1988. Diabetes, clinical pharmacy and therapeutics. William & Wilkins. 4, p. 176). Diabetes is characterized by hormonal imbalances such as insulin, including glucagon and glucocorticoids, and characteristic symptoms such as hyperglycemia and diabetes due to abnormal physiological metabolic control functions such as carbohydrates, protein, lipid and electrolyte metabolism. In addition, as a complication of diabetes, vascular disorders, neurological disorders and infectious diseases are mainly shown. α-glucosidase is an enzyme involved in the end of carbohydrate digestion. Substances with inhibitory activity inhibit the absorption of carbohydrates, which can suppress rapid blood sugar rise after eating, resulting in diabetes and obesity. Used to control patients (Int. J. Obes. 11 (Supple 2): 28, 1987). The most common oral antidiabetic agent for α-glucosidase is acarbose (Digestion. 23: 232-238, 1982), but it causes hypoglycemic shock, gasoline and gastrointestinal disorders. There is a problem that causes. Therefore, it is necessary to develop a hypoglycemic agent with less side effects and safer.

Soybean leaves were significantly reduced in isoflavones and flavonoids compared to soybeans, while containing higher kaempferol glycosides (Biomed. Pharmacother. 56: 289-295, 2002). In addition, mature soybean leaves separated from pterocarpane compounds such as coumestrol, glycyceollin derivatives, isotrifoliol, and phaseol. Glucosidase and LDL antioxidant activity have been reported (J. Agric. Food Chem., 54: 2057-2063, 2006; Food Chemistry, 126: 1057-1063, 2011; J. Agric. Food Chem., 59: 12683-12690, 2011). However, the literature reported by separating the fairovide compound from soybean leaves has not been found. In addition, the virucidal effect of the fairovide compound isolated from Cactus (Opuntia ficus indica) (J. Nat. Med., 65: 229-233, 2011), fairpo separated from Typhonium flagelliforme Anticancer Activity of Bide Compounds (J. Ethnopharmacology, 127: 486-494, 2010) and Antioxidant Activity of Fairpoid Compounds Isolated from Enteromorpha prolifera (Food Chemistry, 127: 999-1006, 2011) Although known, the inhibitory activity of (alpha) -glucosidase of a fairovide type compound has not been reported.

Accordingly, the inventors of the present invention, while searching for a preventive and therapeutic agent for metabolic diseases including diabetes or their complications, found that the fairovide compound obtained from the soybean leaf extract has an inhibitory activity of α-glucosidase. After confirming, the fairovide-based compound was confirmed that it can be usefully used as a composition for the prevention and treatment of metabolic diseases including diabetes or their complications and completed the present invention.

It is an object of the present invention to provide a pharmaceutical composition for the prevention and treatment of metabolic diseases or complications thereof, which contains a phenophorbide-based compound, a tautomer compound thereof, or a pharmaceutically acceptable salt thereof as an active ingredient. It is.

Another object of the present invention is to provide a health food composition for the prevention and improvement of metabolic diseases or complications thereof containing a fairovide compound, a tautomer compound thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

Still another object of the present invention is to provide a method for preparing the fairovide compound.

In order to achieve the above object, the present invention is for the prevention and treatment of metabolic diseases or complications thereof containing a phenophorbide-based compound, tautomer compounds thereof or a pharmaceutically acceptable salt thereof as an active ingredient It provides a pharmaceutical composition.

In another aspect, the present invention provides a composition for health food for the prevention and improvement of metabolic diseases or complications thereof containing a fairovide compound, a tautomer compound thereof or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition,

1) fermenting soybean leaves;

2) adding an extraction solvent to the fermented soybean leaves of step 1) to obtain a soybean leaf extract; And

3) provides a method of mass-producing a fairovide compound comprising the step of separating and purifying the compound by performing column chromatography using an organic solvent as an extraction solvent for the fraction obtained in step 2).

Pheophorbide compounds according to the present invention effectively inhibit the activity of α-glucosidase and have little toxicity, thus preventing and treating metabolic diseases including diabetes or their complications. It can be useful.

1 is a graph of a proton nuclear magnetic resonance spectrum ( ¹ H NMR spectrum) of Compound 1. FIG.
FIG. 2 is a graph of a ¹³ C NMR spectrum of Compound 1; FIG.
Figure 3 is a graph of the proton-proton two-dimensional Cozy nuclear magnetic resonance spectrum ( ¹ H- ¹ H COSY spectrum spectrum) of Compound 1.
4 is a graph of the heteronuclear multiple quantum correlation spectroscopy spectrum (HMQC spectrum) of Compound 1
FIG. 5 is a graph of the heteronuclear multiple bond correlation spectroscopy spectrum of Compound 1. FIG.
6 is a diagram showing a two-dimensional nuclear magnetic resonance correlation of Compound 1.
7 is a diagram showing the structure of compound 1. FIG.
8 is a graph of the electrospray ionization mass spectrum (ESI-mass spectrum) in the negative mode of compound 1.
9 is a graph of a proton nuclear magnetic resonance spectrum ( ¹ H NMR spectrum) in chloroform (CHCl ₃ ) of compound 1. FIG.
FIG. 10 is a graph of a carbon nuclear magnetic resonance spectrum ( ¹³ C NMR spectrum) in chloroform (CHCl ₃ ) of compound 1.
11 is a graph of the proton nuclear magnetic resonance spectrum of Compound 2 ⁽¹ H NMR spectrum).
12 is a graph related to high performance liquid chromatography (HPLC) profiles at 254 nm of compound 1
FIG. 13 is a graph of high performance liquid chromatography (HPLC) profile at 420 nm of compound 1.
14 is a graph relating to the UV / VIS spectrum of fairovide a.

Hereinafter, the term of this invention is demonstrated.

As used herein, the term "prophylactic " refers to any action that inhibits the onset or delays the onset of the administration of the composition. In the present invention, "improvement" or "treatment" means any action in which administration of the composition improves or alters the symptoms of the disease.

In the present invention, "administration" means providing a predetermined substance to a patient in any suitable manner, and the administration route of the composition of the present invention may be oral or parenteral ≪ / RTI > The composition may also be administered by any device capable of transferring the active agent to the target cell.

Hereinafter, the present invention will be described in detail.

The present invention is a pharmaceutical composition for the prevention and treatment of metabolic diseases or complications thereof comprising a phenophorbide-based compound represented by the following formula (1), a tautomer thereof or a pharmaceutically acceptable salt thereof as an active ingredient Provides:

In the above formula, R ¹ , R ² and R ³ is H, C ₁ ~ C ₄ linear or branched alkyl, carboxyl, C ₁ ~ C ₄ alkoxycarbonyl, C ₁ ~ C ₄ alkoxycarbonyl C ₁ ~ Any one selected from the group consisting of C ₄ alkyl, amino group, amino C ₁ -C ₄ alkyl, and oxo (═O),

R ⁴ is selected from the group consisting of H, hydroxy, oxo (═O), C ₁ -C ₄ straight or branched alkyl, C ₁ -C ₄ straight or branched alkoxy, amino and amino C ₁ -C ₄ alkyl Which one.

Preferably, the fairovide compound is preferably represented by the following [Formula 2], but is not limited thereto:

The fairovide-based compound preferably has an inhibitory effect of α-glucosidase (α-glucosidase), but is not limited thereto.

The metabolic disease is preferably selected from the group consisting of diabetes, hyperlipidemia, arteriosclerosis, fatty liver and obesity, but is not limited thereto.

The complications are selected from the group consisting of coronary artery disease, angina pectoris, carotid disease, stroke, cerebral atherosclerosis, hypercholesterolemia, cholesterol stones, hypertriglyceridemia, hypertension, cataracts, kidney disease, neuropathy, chronic inflammatory disorders and infectious diseases But is not limited thereto.

The fairovide compound and tautomers thereof are preferably prepared in the following steps, but are not limited thereto.

1) water bean leaves, C ₁ To Adding a C ₄ alcohol or a mixture thereof to obtain a soybean leaf extract;

2) obtaining the fraction by sequentially fractionating the soybean leaf extract obtained in step 1) using water, dichloromethane and ethyl acetate; And

3) separating and purifying the compound by performing column chromatography on the ethyl acetate fraction obtained in step 2) using an organic solvent as an effluent solvent.

Hereinafter, the production method of the present invention will be described step by step.

Step 1) is a step of obtaining soybean leaf extract by adding water, C ₁ ~ C ₄ alcohol or a mixture thereof.

In the above step, the method for obtaining the canola extract of the present invention comprises the steps of dividing the dried canned leaves into an extraction container, adding water, a C ₁ to C ₄ alcohol or a mixture thereof to extract at room temperature or warming to obtain an extract. The above procedure may be repeated several times in order to increase the extraction efficiency of the bean leaves. Thereafter, the extract is left for a predetermined time and then filtered with a filter paper to prepare an ethanol extract.

Step 2) is a step of obtaining the fraction by sequentially fractionating the soybean leaf extract obtained in step 1) using water, dichloromethane and ethyl acetate.

Specifically, the soybean leaf extract obtained in the above step is suspended in water, and then sequentially fractionated using dichloromethane and ethyl acetate as extraction solvents to obtain a dichloromethane fraction, an ethyl acetate fraction and a water fraction.

In step 3), the ethyl acetate fraction obtained in step 2) is subjected to column chromatography using an organic solvent as an outflow solvent to separate and purify the compound.

Specifically, the ethyl acetate fraction of the fractions obtained from step 2) was chloroform / methanol = 100: 0 ~ 9: 1 concentration gradient using chloroform / methanol as the effluent solvent to perform silica gel column chromatography to perform 10 fractions ( A to J), and the E fraction (chloroform: methanol = 24: 1) was gradientd to chloroform / methanol = 100: 0 to 24: 1, and silica gel column chromatography was performed to obtain 8 (FE1 to FE8). After fractionation of the fractions of the eight fractions, fractions of FE5 (chloroform: methanol = 97: 3) was gradient gradient of chloroform / methanol = 100: 0 ~ 24: 1 to perform silica gel column chromatography four ( Fractions of FE5-1 to FE5-4), and then the fractions of FE5-1 and FE5-2 were combined and water containing acetonitrile / 0.1% acetic acid = 5: 95-100: 0 C18-reverse high performance column chromatography (HPLC) Performed to remove the compounds (1) and its tautomers represented by the following formula (2), it can be purified.

In addition, the fairovide compound and tautomers thereof may be prepared by the following steps:

1) adding ethyl acetate to the soybean leaves to obtain a soybean leaf ethyl acetate extract; And

2) separating and purifying the compound by performing column chromatography using chloroform / methanol as the effluent solvent of the soybean leaf extract obtained in step 1 above.

Hereinafter, the production method of the present invention will be described step by step.

Step 1) is a step of extracting the soybean leaves with ethyl acetate.

In the above step, the method for obtaining ethyl acetate extract is chopped dried soybean leaves into an extraction container, put ethyl acetate and extracted at room temperature to obtain an extract. The process may be repeated several times to increase the extraction efficiency of the soybean leaves. Thereafter, the extract is left for a predetermined time and then filtered through a filter paper to prepare ethyl acetate extract.

In step 2), the ethyl acetate extract obtained in step 1) is subjected to column chromatography using an organic solvent as an effluent solvent to separate and purify the compound.

The step may be carried out in the same manner as step 3) of the above-described manufacturing method.

In a specific embodiment of the present invention, the present inventors prepared soybean leaf extract to separate the pheophobide-based compound from the soybean leaves, and then subjected to column chromatography to separate the compound 1 and tautomers thereof represented by the formula (2). .

In addition, the present inventors conducted nuclear magnetic resonance (NMR) and mass spectrometry for structural analysis of the separated compound, and the compound confirmed the pairpoide a and tautomer thereof represented by Chemical Formula 2 (Fig. 1 to 11).

In addition, the present inventors performed the α-glucosidase inhibitory activity measurement to verify the activity related to the metabolic disease of the fairovide compound represented by the formula (2) and tautomers thereof, the fairovide compound is α- It glucosidase during appeared to Days activity that is excellent in a concentration of IC ₅₀ values for inhibition of 50%, in particular, each of the IC ₅₀ value of the compound represented by the compound and its tautomer (tautomer) of the formula 2 76.4 μM (45.2 μg / ml) and 86.1 μM (51.0 μg / ml) showed high inhibitory activity of α-glucosidase activity (see Table 1).

In addition, the present inventors conducted a toxicity test by orally administering the fairovide compound of the present invention to mice, the fairovide compound is 50% lethal dose (LD ₅₀ ) by oral toxicity test at least 1000 mg / It confirmed that it was more than kg.

Therefore, the fairovide compound of the present invention effectively inhibits α-glucosidase activity and has little toxicity, and thus may be usefully used as a pharmaceutical composition for preventing and treating metabolic diseases including diabetes or its complications. .

The present invention includes not only the fairovide compound represented by the formula (2) and tautomers thereof, but also pharmaceutically acceptable salts thereof, and possible solvates, hydrates, racemates, or stereoisomers which can be prepared therefrom. do.

The fairovide compound represented by the formula (2) of the present invention and tautomers thereof may be used in the form of a pharmaceutically acceptable salt, and the salt may be an acid addition salt formed by a pharmaceutically acceptable free acid. This is useful. Acid addition salts include those derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates, Dioleate, aromatic acid, aliphatic and aromatic sulfonic acids. Such pharmaceutically innocuous salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate chloride, bromide, Butyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, succinate, maleic anhydride, maleic anhydride, , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzene sulfonate, toluene sulfonate, chlorobenzene sulfide Sulfonate, methanesulfonate, propanesulfonate, naphthalene-1-sulphonate, naphthalene-1-sulphonate, , Naphthalene-2-sulfonate or mandelate.

The acid addition salt according to the present invention is a conventional method, for example, at least one compound of the fairovide compound represented by the formula (2) and tautomers thereof is dissolved in an excess of an aqueous acid solution, and the salt is a water miscible organic solvent. For example, it can be prepared by precipitation using methanol, ethanol, acetone or acetonitrile.

In addition, the same amount of the fairovide compound represented by the formula (2) or a tautomer thereof, and an aqueous acid solution or alcohol may be heated, and then the mixture is evaporated to dryness or a precipitated salt may be produced by suction filtration.

In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt. The corresponding silver salt is also obtained by reacting an alkali metal or alkaline earth metal salt with a suitable salt (such as silver nitrate).

When formulating the composition, it is prepared using commonly used diluents or excipients, such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants.

Solid preparations for oral administration include tablets, patients, powders, granules, capsules, troches, and the like, which solid preparations contain at least one of the fairovide compounds represented by Formula 2 of the present invention and tautomers thereof. One or more excipients, for example, starch, calcium carbonate, sucrose or lactose, gelatin and the like are mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions or syrups. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like are included in addition to commonly used simple diluents such as water and liquid paraffin. .

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like.

Examples of the non-aqueous solvent and suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As a base for suppositories, witepsol, macrogol, tween 61, cacao paper, laurin, glycerol, gelatin and the like can be used.

The composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will depend on the type of disease, severity, , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including co-administered drugs, and other factors well known in the medical arts. The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the compound according to the present invention may vary depending on the age, sex, and weight of the patient, and in general, 0.1 mg to 100 mg, preferably 0.5 mg to 10 mg per 1 kg of body weight is administered daily or every other day. Or divided into 1 to 3 times a day. However, the dosage may be varied depending on the route of administration, the severity of obesity, sex, weight, age, etc. Therefore, the dosage is not limited to the scope of the present invention by any means.

In addition, the present invention is a health for the prevention and improvement of metabolic diseases or complications thereof containing a pheophorbide-based compound represented by the following formula (1), a tautomer thereof or a pharmaceutically acceptable salt thereof as an active ingredient Provided are food compositions:

[Chemical Formula 1]

.

.

In the above formula, R ¹ , R ² and R ³ is H, C ₁ ~ C ₄ linear or branched alkyl, carboxyl, C ₁ ~ C ₄ alkoxycarbonyl, C ₁ ~ C ₄ alkoxycarbonyl C ₁ ~ Any one selected from the group consisting of C ₄ alkyl, amino group, amino C ₁ -C ₄ alkyl, and oxo (═O),

R ⁴ is selected from the group consisting of H, hydroxy, oxo (═O), C ₁ -C ₄ straight or branched alkyl, C ₁ -C ₄ straight or branched alkoxy, amino and amino C ₁ -C ₄ alkyl Which one.

Preferably, the fairovide compound is preferably represented by the following [Formula 2], but is not limited thereto:

 (2)

.

.

In a specific embodiment of the present invention, the present inventors performed a-glucosidase inhibitory activity measurement to verify the activity related to the metabolic disease of the fairovide-based compound represented by the formula (2) and tautomers thereof, carbide-based compound appeared to be excellent in a concentration of IC ₅₀ for inhibiting 50% of the activity during Days α- glucosidase value, in particular, the IC ₅₀ value of the compound represented by the compound and its tautomer (tautomer) represented by the formula (2) 76.4 μM (45.2 μg / ml) and 86.1 μM (51.0 μg / ml), respectively, showed high α-glucosidase activity inhibitory activity (see Table 1).

In addition, the present inventors conducted a toxicity test by orally administering the fairovide compound of the present invention to mice, the fairovide compound is 50% lethal dose (LD ₅₀ ) by oral toxicity test at least 1000 mg / It confirmed that it was more than kg.

Therefore, the fairovide compound of the present invention effectively inhibits α-glucosidase activity and has little toxicity, and thus can be usefully used as a composition for health food for preventing or improving metabolic diseases including diabetes or its complications. have.

There is no restriction | limiting in particular in the kind of food to which the fairovide type compound of this invention is added. Examples of the foods to which the above substances can be added include dairy products including dairy products, meat, sausage, bread, biscuits, rice cakes, chocolate, candies, snacks, confectionery, pizza, ramen and other noodles, gums, ice cream, Beverages, alcoholic beverages and vitamin complexes, dairy products, and dairy products, all of which include health functional foods in a conventional sense.

The fairovide compound of the present invention may be added to a food as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (for prevention or improvement). Generally, the amount of the compound in the health functional food may be 0.1 to 90 parts by weight based on the total weight of the food. However, in the case of long-term intake intended for health and hygiene purposes or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

When the health food composition according to the present invention is a beverage composition, there are no particular restrictions on other components other than those containing the above-mentioned compounds as essential components in the indicated ratios, and various flavors or natural carbohydrates, ≪ / RTI > Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 10 g per 100 of the composition of the present invention.

In addition, the health food composition according to the present invention can be used as a nutritional supplement, a vitamin, a mineral (electrolyte), a flavoring agent such as a synthetic flavor agent and a natural flavor agent, a coloring agent and a thickening agent (cheese, chocolate etc.) Alginic acid and its salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks and the like. Others may contain pulp for the production of natural fruit juices and fruit juice beverages and vegetable beverages.

These components may be used independently or in combination. The proportion of such additives is not limited, but is generally selected from 0.1 to about 20 parts by weight per 100 parts by weight of the fairovide compound.

In addition,

1) fermenting soybean leaves;

2) adding an extraction solvent to the fermented soybean leaves of step 1) to obtain a soybean leaf extract; And

3) provides a method of mass-producing a fairovide compound comprising the step of separating and purifying the compound by performing column chromatography using an organic solvent as an extraction solvent for the fraction obtained in step 2).

In a specific embodiment of the present invention, the inventors confirmed the content of the fairpoide-based compound by the aging or fermentation of soybean leaves, hot air dried soybean leaves after ripening according to the present invention 70% ethanol extract of the hot air dried soybean leaves are not aged It can be seen that the peak area of the fairpoide a is significantly increased compared to the 70% ethanol extract of, and this result is consistent with the increase of α-glucosidase activity inhibitory ability.

Therefore, it was confirmed that the fair fobide-based compound according to the present invention increases its content through aging or fermentation using microorganisms of soybean leaves (FIGS. 12 to 14).

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

However, the following Examples and Experiments are merely illustrative of the contents of the present invention, and the scope of the present invention is not limited by Examples and Experimental Examples.

< Example  1> from bean leaves Fair forbidden system  Separation of compounds

<1-1> Preparation of Soybean Leaf Extract

Soybean Leaf ( Glycine) max In order to isolate the fairovide compound from the leaf, soybeans were sown in Jeungpyeong, Chungcheongnam-do, and the soybeans harvested after 90 days of growth were dried in the shade. 300 g of dried and pulverized soybean leaves were added with 3 L of ethyl acetate and extracted at room temperature for 3 days. Then, the primary ethyl acetate soluble part was recovered using filter paper, and the bean leaf residue was extracted and filtered again in the same manner. The acetate solubles were recovered. The primary and secondary ethyl acetate soluble parts were combined and concentrated under reduced pressure to obtain 13 g of ethyl acetate extract.

<1-2> Fair forbidden system  Separation of compounds

The ethyl acetate extract (13 g) obtained in Example <1-1> was isolated using a purification system (SP4 HPFC, biotage, Sweden, Uppsala). The silica gel column used was FLASH 75 + M (Biotage SNAP cartrige, 75 mm X 150 mm), the flow rate was 75 ml / min, and the separation fraction was detected under UV 254 nm using chloroform-methanol mixed solution as the solvent. Chloroform / methanol = 100: 0 ~ 9: 1, eluted with a concentration gradient, fractionated by 1 minute (75 ml) per tube, separated into a total of 100 tubes, and thin layer chromatography and UV absorbance were measured. The tubes containing compounds with similar Rf values were combined and fractionated into 10 fractions (A-J).

The fraction E (chloroform: methanol = 24: 1, 2.8 g) was gradientd to chloroform / methanol = 100: 0 to 24: 1 and subjected to silica gel column chromatography to obtain 8 fractions (FE1 to FE8) again. In addition, the fractions of FE5 (chloroform: methanol = 97: 3, 862 mg) were gradient-graded to chloroform / methanol = 100: 0 to 24: 1 to perform silica gel column chromatography (FE5-1 to FE5-4). And fractions of FE5-1 and FE5-2 (172 mg) were combined to obtain acetonitrile / 0.1% acetic acid and the mobile phase (0.1% acetic acid). Solvent A) and acetonitrile (solvent B); a linear gradient elution program; 5 to 80% B for 0 to 20 min, 80 to 95% B for 20 to 35 min, 95 to 100% B for 35 to 40 min ; 100% B for 40-45 min; 100-5% B for 45-55 min) using C18-reverse high performance column chromatography (HPLC, RP-C18, Cosmosil 5C18-MS-II, 10 x 250 mm, 5 μm, 3 ml / min; 254 nm) 1 was obtained;; (2 mg Rt = 35.7 bun) (Rt = 33.8 bun 8 mg) and compound 2.

< Example  2> separated from soybean leaves Fair forbidden system  Structural Analysis of Compounds

Nuclear magnetic resonance (NMR) analysis and mass spectrometry were performed for structural analysis of the compound separated in Example 1.

<2-1> proton nuclear magnetic resonance spectrum ( ^One H NMR spectrum Measurement and interpretation

For the structural analysis of Compound 1 isolated in Example 1, a proton nuclear magnetic resonance spectrum ( ¹ H NMR spectrum) was measured, and six aromatic or olefinic groups were found between 9.20 and 6.0 ppm. ) Proton signals were identified, two methine protons at 4.48 and 4.11 ppm, four methyl peaks at 3.89, 3.53, 3.28 and 2.89 ppm, two between 2.7 and 2.1 ppm Two methyl peaks were identified at 1, equivalent methylene peak of 1.80, 1.51 ppm (FIG. 1).

<2-2> carbon nuclear magnetic resonance spectrum ( ¹³ C NMR spectrum Measurement and interpretation

For the structural analysis of Compound 1 isolated in Example 1, a carbon nuclear magnetic resonance spectrum ( ¹³ C NMR spectrum) was measured, and a plurality of carbonyl carbons and aromatics were found between 191 and 93 ppm. 23 sp ² due to aromatic and olefinic carbon 11 sp ³ carbon peaks due to carbon, methine, methylene, and methyl carbon were identified (FIG. 2).

<2-3> proton-proton two-dimensional Cozy spectrum ( ^One H- ^One H COZY spectrum Measurement and interpretation

In order to determine the partial structure of the compound 1 separated by the <Example 1> The proton-proton two-dimensional spectrum koji ⁽¹ H- ¹ H COSY spectrum) analysis was measured (Fig. 3).

As a result, as shown in Fig. 6, the presence of _three substructures, CH ₂ = CH-, CH ₃ -CH-CH-CH ₂ -CH _2- , and CH ₃ -CH ₂ -was identified (Fig. 6).

<2-4> heteronuclear multiple quantum correlation spectroscopy spectrum ( HMQC spectrum ) And heteronuclear multiple bond correlation spectroscopy spectra ( HMBC spectrum Measurement and interpretation

For structural analysis of Compound 1 isolated in <Example 1>, heteronuclear multi quantum correlation spectroscopy (HMQC spectrum) was measured and analyzed, and the result was confirmed that the proton-bearing carbon ( ¹ J _{C -H} ) ( 4).

In addition, the heteronuclear multiple bond correlation spectroscopy (HMBC spectrum) measured and interpreted, the methyl group is a strong ring current effect (anisotropic effect) from the long-range correlation observed on sp2 carbon from 3.53, 3.28, 2.89 ppm methyl proton ) Was confirmed to receive (Fig. 5). These phenomena appearing in the chlorophyll-based compound could be inferred that the compound is also a chlorophyll-based compound. Therefore, the cross-peaks shown in the HMBC spectrum were compared with the chlorophyll structure, and the results are shown in FIG. 6. On the basis of this, the chemical structure of Compound 1 represented by the following [Formula 2] was confirmed. As a result of performing a database search based on the chemical structure, the compound was found to be a pheophorbide a. It was.

(2)

.

.

<2-5> mass spectrum ( Mass spectrum Measurement and interpretation

From the nuclear magnetic resonance (NMR) spectroscopic analysis, the chemical structure of Compound 1 was identified as a pair of paired pairs (Peophorbide a), and then mass (mass) analysis was performed for identification thereof.

Specifically, the electrospray ionization mass (ESI-mass) was measured in the negative mode, and the molecular weight was confirmed. That is, [MH]-was observed at m / z 591 to confirm that the molecular weight was 592, which was confirmed to correspond exactly with the chemical structure of NMR spectroscopy results (FIG. 8).

<2-6> nuclear magnetic resonance spectrum ( NMR spectrum Measurement and interpretation

For the structural analysis of Compound 2 isolated in <Example 1>, CDCl ₃ : Proton nuclear magnetic resonance spectrum ( ¹ H NMR spectrum) was measured by dissolving in a mixed solvent of CD ₃ OD.

As a result, as shown in FIG. 11, Compound 2 was identical to Compound 1, but 2.89 ppm of methyl group moved to the high magnetic field, and 8.96 ppm of methine protons moved to the low magnetic field. Since the compound was confirmed to be a fairovide A tautomer (tautomer) produced by tautomerism of the fairpoide a (Fig. 11).

< Experimental Example  1> Fair forbidden system   The α- Glucosidase (? glucosidase ) activation Low performance  Measure

Fairivide system separated in <Example 1> The following experiments were conducted to determine the effect on α-glucosidase activity essential for carbohydrate metabolism of the compound.

The inhibitory activity against? -glucosidase was partially modified by the nitrophenol method of Kato et al. (J. Med. Chem. 48: 2036-2044, 2005). Hydrolysis of p-nitrophenyl-D-glucopyranoside (Sigma-Aldrich) as a substrate by α-glucosidase (EC 3.2.1.20, Baker Yeast) occurred Accumulation of the resulting chromophore was measured by absorbance.

Specifically, 50 μl of buffer solution (70 mM calcium phosphate, pH 6.8) in a 96-well plate (NUNC ^™ ), 50 μl of sample compound dissolved in 50% DMSO, 50 μl of α-glucosidase (0.1 unit / ml) After adding the substrate (5 mM, p-nitrophenyl-D-glucopyranoside) and finally reacting at 37 ° C. for 30 minutes, 2 M NaOH was added to terminate the reaction. The amount of the resulting chromophore was measured at 405 nm using a microplate reader (BIORAD) and the inhibitory activity was calculated and the results are shown in Table 1 below.

Fairovide Compound α-glucosidase inhibitory activity (IC ₅₀ , μM) (2) 76.4 (or 45.2 μg / ml) Tautomers of Formula 2 86.1 (or 51.0 μg / ml)

As shown in Table 1, the fairovide compound according to the present invention was found to have a superior IC ₅₀ value, a concentration that inhibits α-glucosidase activity by 50%, in particular, Compound 1 and The IC ₅₀ values of the tautomers thereof were 76.4 μM (45.2 μg / ml) and 86.1 μM (51.0 μg / ml), respectively, and confirmed high α-glucosidase activity inhibition (Table 1).

< Experimental Example  2> by aging or fermentation Fair forbidden system  Increased content of compounds

The following experiment was carried out to determine the effect of increasing the content and activity according to the ripening of soybean leaves of the fairovide compound isolated in <Example 1>.

Harvested soybean leaves were sealed in a bag and left to stand in the sun for 2 days in order to obtain mature soybean leaves. Generally, aging is a chemical that slowly ferments or forms colloidal particles when the material is left for a long time at a moderate temperature. ) Refers to the change, and in the present invention, fermented soybean leaves using microorganisms (fungus, etc.) contaminated in the natural space, and for artificial fermentation is not limited to microorganisms contaminated in the natural space, edible lactic acid bacteria, yeast, Duruk, Bacillus, etc. can be used. After ripening soybean leaves were extracted in the same manner as in <Example 1>. The weight of the 70% ethanol extract, 95% ethanol extract, or ethacetate extract obtained from the aged soybean leaves were obtained 30.0 mg, 19.0 mg, 8.5 mg of the soybean leaf extract, respectively. Therefore, the weight of aged soybean extract was the same or slightly increased (Table 2).

Next, the α-glucosidase inhibitory activity of each extract was measured in the same manner as in Experimental Example 1, and the results are shown in Table 2 below.

Bean Leaf Extract Soy Leaf Extract Drying Weight
(mg / 100 mg soybean leaves) α-glucosidase inhibitory activity (%) Hot air dried bean leaves 70% Ethanol Extract 30.5 ± 0.1 21.9 ± 3.1 95% ethanol extract 16.3 ± 0.2 18.7 ± 1.5 Ethyl acetate extract 6.0 ± 0.0 22.1 ± 2.4 After ripening
Hot air dried bean leaves 70% Ethanol Extract 30.0 ± 0.0 35.1 ± 0.4 95% ethanol extract 19.0 ± 0.1 37.7 ± 3.5 Ethyl acetate extract 8.5 ± 0.1 34.9 ± 1.1

As shown in Table 2, the hot-air dried soybean leaves after fermentation according to the present invention are significantly increased in all of 70% ethanol extract, 95% ethanol extract, or ethyl acetate extract compared to hot air dried soybean leaves. Days activity inhibition was shown.

In order to support these results, each extract was analyzed by high performance liquid chromatography (HPLC) to determine the change in the content of the fairovide compound.

Specifically, Shimadzu HPLC system (Pump: Shimadzu LC-10A vp with binary pump; Detector: Shimadzu SPD-M10A vp PDA detector, 254 nm & 420 nm; Auto injector: Shimadzu SIL-10A vp) was used for HPLC analysis. , HPLC column was used Brownlee SPP C18-MS-II (4.6 x 50 mm, 2.7 μm). Water containing acetonitrile / 0.1% acetic acid was transferred to a mobile phase {water containing 0.1% acetic acid (solvent A) and acetonitrile (solvent B); a linear gradient elution program; 580% B for 0-20 min, 80-95% B for 20-35 min, 95-100% B for 35-40 min; 100% B for 40-45 min; 100 ~ 5% B for 45 ~ 55 min} was used to separate and elute at a flow rate of 1.8 ml / min conditions are shown in Figures 12 to 14.

As a result, as shown in Figures 12 to 14, the peak of the pairpoide a was confirmed in 20.2 components, 70% of hot air dried soybean leaves after ripening according to the present invention 70% ethanol extract of hot air dried soybean leaves after ripening Compared to the% ethanol extract, it can be seen that the peak area of the fairpoide a is significantly increased, and this result is consistent with the increase of α-glucosidase activity inhibitory ability.

Therefore, it was confirmed that the fair fobide-based compound according to the present invention increases its content through fermentation or fermentation using microorganisms of soybean leaves (FIGS. 12 to 14).

< Experimental Example  3> ICR  Acute toxicity test for oral administration to mice

In order to determine the acute toxicity of the fairovide compound included in the composition according to the present invention, the following experiment was performed.

<3-1> ICR  Manufacture of mice

12 females and 12 males (3 males and 3 females each) at 4 weeks of age for specific pathogens-free ICR mice were kept in an animal room with a temperature of 22 ± 3 ° C, a humidity of 55 ± 10%, and illumination of 12 L / 12D. Breeding. Mice were purified for a week before being used in the experiment. Feeds for experimental animals (Cheil Jedang Co., Ltd., mice and rats) and drinking water were sterilized and fed freely.

<3-2> Drug Administration

The fairovide compound represented by Formula 2 obtained in <Example 1> was prepared at a concentration of 50 mg / ml using 0.5% Tween 80 as a solvent, and then 0.2 ml (500 mg / kg) per 20 g of mouse body weight. 0.4 ml (1,000 mg / kg) were administered orally.

Samples were administered orally only once and observed for side effects or lethality for 7 days after administration. That is, on the day of administration, changes in general symptoms and the presence of dead animals were observed at least once in the morning, at least once every afternoon from 1 hour, 4 hours, 8 hours, 12 hours, and the next day after the administration. In addition, on the 7th day of administration, animals were killed and dissected, and the internal organs were visually examined. Changes in body weight were measured at daily intervals from the day of administration to observe the weight loss phenomenon of the animals caused by the fairovide compound.

As a result, no significant clinical symptoms were observed in all mice treated with the test substance, no mice died, and no toxic changes were observed in weight changes, blood tests, blood biochemical tests, and autopsy findings. Therefore, the fairovide compound represented by Formula 2 did not show a toxic change to 1000 mg / kg in all mice, and was determined to be a safe substance having an oral administration minimum dose (LD ₅₀ ) of at least 1,000 mg / kg or more.

Therefore, the fairovide compound according to the present invention can effectively be used as a composition for preventing or treating metabolic diseases or complications thereof, including diabetes, in which side effects are reduced because they effectively inhibit α-glucosidase activity.

< Manufacturing example  1> Manufacture of Pharmaceuticals

One. Sanje  Produce

500 ng of fairovide compound of the present invention

1 g lactose

The above components were mixed and packed in airtight bags to prepare powders.

2. Preparation of tablets

500 ng of fairovide compound of the present invention

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

3. Preparation of Capsule

500 ng of fairovide compound of the present invention

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.

4. Preparation of injections

500 ng of fairovide compound of the present invention

Mannitol 180 mg

Na2HPO42H2O 26 mg

Distilled water 2974 mg

According to a conventional method for preparing an injection, an injection was prepared by containing the above components in the contents shown.

< Manufacturing example  2> Manufacture of health food

1. Manufacture of health food

500 ng of fairovide compound of the present invention

Vitamin mixture quantity

70 [mu] g of vitamin A acetate

Vitamin E 1.0 mg

0.13 mg of vitamin

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

0.2 [mu] g vitamin B12

Vitamin C 10 mg

Biotin 10 μg

Nicotinamide 1.7 mg

Folic acid 50 mg

Calcium Pantothenate 0.5 mg

Mineral mixture quantity

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

15 mg potassium monophosphate

Dicalcium Phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride 24.8 mg

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

2. Manufacture of health drinks

500 ng of fairovide compound of the present invention

Citric acid 1000 mg

100 g of oligosaccharide

Plum concentrate 2 g

Taurine 1 g

Add 900 ml of purified water

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

Although the composition ratio is 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 (8)

A pharmaceutical composition for preventing and treating metabolic diseases or complications thereof, which comprises a phenophorbide-based compound represented by Formula 1, a tautomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient:
[Chemical Formula 1]

(In Formula 1, R ¹ , R ² and R ³ is H, C ₁ ~ C ₄ straight or branched chain alkyl, carboxyl, C ₁ ~ C ₄ alkoxycarbonyl, C ₁ ~ C ₄ alkoxycarbonyl C Any one selected from the group consisting of ₁ to C ₄ alkyl, an amino group, amino C ₁ to C ₄ alkyl, and oxo (═O),
R ⁴ is selected from the group consisting of H, hydroxy, oxo (═O), C ₁ -C ₄ straight or branched alkyl, C ₁ -C ₄ straight or branched alkoxy, amino and amino C ₁ -C ₄ alkyl Which one).
The pharmaceutical composition for preventing and treating metabolic diseases or complications thereof according to claim 1, wherein the fairovide compound is represented by the following Chemical Formula 2:
(2)

.
[Claim 2] The pharmaceutical composition for preventing and treating metabolic diseases or complications thereof according to claim 1, wherein the fairovide compound has an inhibitory effect of α-glucosidase.
The method of claim 1, wherein the metabolic disease is diabetes, hyperlipidemia, arteriosclerosis, fatty liver and obesity, characterized in that any one selected from the group consisting of obesity metabolic disease or a pharmaceutical composition for the prevention and treatment of complications thereof.
The method of claim 1, wherein the complications are coronary artery disease, angina pectoris, carotid artery disease, stroke, cerebral arteriosclerosis, hypercholesterolemia, cholesterol stones, hypertriglyceridemia, hypertension, cataracts, kidney disease, neuropathy, chronic inflammatory disorders and infectious diseases A pharmaceutical composition for preventing and treating metabolic diseases or complications thereof, characterized in that any one selected from the group consisting of.
A composition for health food for preventing and improving metabolic diseases or complications thereof, which comprises a fairovide compound represented by the following Formula 1, a tautomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient:
[Chemical Formula 1]

(In Formula 1, R ¹ , R ² and R ³ is H, C ₁ ~ C ₄ straight or branched chain alkyl, carboxyl, C ₁ ~ C ₄ alkoxycarbonyl, C ₁ ~ C ₄ alkoxycarbonyl C Any one selected from the group consisting of ₁ to C ₄ alkyl, an amino group, amino C ₁ to C ₄ alkyl, and oxo (═O),
R ⁴ is selected from the group consisting of H, hydroxy, oxo (═O), C ₁ -C ₄ straight or branched alkyl, C ₁ -C ₄ straight or branched alkoxy, amino and amino C ₁ -C ₄ alkyl Which one).
[Claim 7] The composition for health food for preventing and improving metabolic diseases or complications thereof according to claim 6, wherein the fairovide compound is represented by the following Chemical Formula 2:
(2)

.
1) fermenting soybean leaves;
2) adding an extraction solvent to the fermented soybean leaves of step 1) to obtain a soybean leaf extract; And
3) A method of mass-producing a fairovide compound comprising the step of separating and purifying the compound by performing column chromatography using an organic solvent as an extraction solvent for the fraction obtained in step 2).

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