KR101638776B1 - 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 pharmaceutical composition for the prevention and treatment of a metabolic disease or a complication thereof comprising an effective ingredient of a parenteral compound, a tautomer thereof or a pharmaceutically acceptable salt thereof, The pfaforbide compound according to the present invention effectively inhibits the activity of? -Glucosidase and has almost no toxicity, so that it can be effectively used for the prevention and treatment of metabolic diseases including diabetes or complications thereof.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for preventing or treating a metabolic disease or a complication thereof, which comprises a parenteral compound or a pharmaceutically acceptable salt thereof as an active ingredient. acceptable salts thereof as an active ingredient}

The present invention relates to a composition for the prophylaxis and treatment of a metabolic disease or a complication thereof comprising an effective ingredient of a pheophorbide-based compound, a tautomer thereof or a pharmaceutically acceptable salt thereof.

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 state because the beta cells of the pancreas are destroyed and does not secrete insulin. Therefore, insulin should be regularly supplied from the outside in order to prevent ketosis and death, And it is called pediatric diabetes. On the other hand, non-insulin dependent diabetes mellitus (type 2 diabetes mellitus), which accounts for more than 90% of all diabetic patients, is a disease caused by a dysfunctional state of insulin, although it has the ability to secrete insulin in the pancreatic beta cells. Diabetes is a hyperglycemic environment in the body that causes high osmotic stress and causes complications such as cataract and kidney disease (Campbell, RK and Steil, CF 1988. Diabetes, clinical pharmacy and therapeutics. William & Wilkins. Diabetes is characterized by hormonal imbalances such as insulin, glucagon and glucocorticoid, which are characteristic of symptoms such as hyperglycemia and diabetes mellitus due to abnormal regulation of physiological metabolism such as carbohydrate, protein, lipid and electrolyte metabolism , And diabetic complications such as vascular disorders, neurological disorders, and infectious diseases. α-glucosidase is an enzyme involved in the end of the digestive process of carbohydrates. Substances having an inhibitory activity of them inhibit the absorption of carbohydrates, thereby suppressing a rapid increase in blood glucose after the meal. Thus, diabetes and obesity It is used to control patients (Int. J. Obes. 11 (Supple 2): 28, 1987). Acarbose (Digestion. 23: 232-238, 1982), which is the most commonly used oral diabetes medicine, is used as an inhibitor of α-glucosidase, but it causes hypoglycemic shock and causes side effects such as gas generation, . Therefore, it is necessary to develop a safe hypoglycemic agent with fewer side effects.

The content of isoflavones and flavonoids in canola leaves was significantly reduced compared to soybeans, while kaempferol glycosides were abundant (Biomed. Pharmacother. 56: 289-295, 2002). The mature bean leaves were also separated from the pterocarpane-based compounds coumestrol, glyceollin derivatives, isotrifoliol and phaseol, and their α- (Agric. Food Chem., 54: 2057-2063, 2006; Food Chemistry, 126: 1057-1063, 2011. J. Agric. Food Chem., 59: 12683-12690, 2011). However, no reports have been found yet on the separation of the phospabide compounds from the soybean leaves. In addition, the virucidal effect of a pair of pivalobide compounds isolated from a cactus (Opuntia ficus indica) (J. Nat. Med., 65: 229-233, 2011), a pair of peptides isolated from Typhonium flagelliforme (Food Chemistry, 127: 999-1006, 2011), which is an anticancer activity of a phybaacide compound (J. Ethnopharmacology, 127: 486-494, 2010) and a pearoboid compound isolated from Enteromorpha prolifera Has been known, but the activity of inhibiting? -Glucosidase of a pearoboid-based compound has not been reported.

Accordingly, the inventors of the present invention have found that when a pharmaceutical composition for prevention and treatment of metabolic diseases including diabetes or complications thereof is investigated, it is confirmed that a fairaboid-based compound obtained from a soybean leaf extract has an inhibitory activity of? -Glucosidase And confirmed that the above-mentioned fairaboid-based compound can be effectively used as a composition for the prevention and treatment of metabolic diseases including diabetes or complications thereof, and completed the present invention.

An object of the present invention is to provide a pharmaceutical composition for the prevention and treatment of a metabolic disease or complication thereof comprising an effective ingredient of a pheophorbide-based compound, a tautomer compound thereof or a pharmaceutically acceptable salt thereof .

Another object of the present invention is to provide a composition for a health food for preventing or ameliorating a metabolic disease or a complication thereof comprising an effective ingredient of a papoboid compound, a tautomer compound thereof or a pharmaceutically acceptable salt thereof.

It is still another object of the present invention to provide a process for producing the above-mentioned fairaboid-based compound.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing and treating a metabolic disease or complication thereof containing an effective ingredient of a pheophorbide-based compound, a tautomer compound thereof or a pharmaceutically acceptable salt thereof A pharmaceutical composition is provided.

The present invention also provides a composition for a health food for preventing or ameliorating a metabolic disease or a complication thereof comprising an effective ingredient of a papoboid compound, a tautomer compound thereof or a pharmaceutically acceptable salt thereof.

In addition,

1) fermenting the soybean leaves;

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

3) performing a column chromatography using an organic solvent as an extraction solvent to separate and purify the compound obtained in the step 2), and then mass-producing the peptide compound.

Since the Pheophorbide compound according to the present invention effectively inhibits the activity of? -Glucosidase and has almost no toxicity, it is useful for the prevention and treatment of metabolic diseases including diabetes or complications thereof It can be useful.

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

Hereinafter, terms of the present invention will be described.

As used herein, the term "prophylactic " refers to any act 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 relates to a pharmaceutical composition for the prevention and treatment of metabolic diseases or complications thereof, comprising a pheophorbide-based compound represented by the following formula (1), a tautomer thereof or a pharmaceutically acceptable salt thereof as an active ingredient Lt; / RTI >

Wherein R ¹ , R ² and R ³ are independently selected from H, C ₁ -C ₄ straight or branched alkyl, carboxyl, C ₁ -C ₄ alkoxycarbonyl, C ₁ -C ₄ alkoxycarbonyl C ₁ - C ₁ -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 linear or branched alkyl, C ₁ -C ₄ straight or branched alkoxy, amino and amino C ₁ -C ₄ alkyl It is either.

Preferably, the pearoboid-based compound is represented by the following formula (2), but is not limited thereto:

The above-mentioned fair-forbidden compound has an inhibitory effect on? -Glucosidase, but is not limited thereto.

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

The complication is selected from the group consisting of coronary artery disease, angina pectoris, carotid artery disease, stroke, cerebral arterial sclerosis, hypercholesterolemia, cholesterol stone, hypertriglyceridemia, hypertension, cataract, renal disease, neurological disorders, chronic inflammatory disorders and infectious diseases But is not limited thereto.

The above-mentioned fair-forbased compound and its tautomer are preferably prepared by the following steps, but are not limited thereto:

1) The canola is water, C ₁ To Adding an alcohol of C ₄ or a mixture thereof to obtain a soybean leaf extract;

2) sequentially fractionating the soybean leaf extract obtained in step 1) using water, dichloromethane and ethyl acetate to obtain fractions; And

3) Separation and purification of the ethyl acetate fraction obtained in step 2) by performing column chromatography using an organic solvent as an eluting solvent.

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

The step 1) is a step of obtaining the soybean leaf extract by adding water, C ₁ -C ₄ alcohol or a mixture thereof to the soybean leaf.

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 allowed to stand for a predetermined period of time and then filtered with a filter paper or the like to produce an ethanol extract.

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

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

Step 3) is a step of separating and purifying the compound by performing column chromatography using the ethyl acetate fraction obtained in the step 2) as an eluting solvent.

Specifically, the ethyl acetate fraction of the fractions obtained from the above step 2) was subjected to silica gel column chromatography using chloroform / methanol as an elution solvent and gradient of chloroform / methanol = 100: 0 to 9: 1 to obtain 10 fractions (FE1 to FE8) was performed by performing silica gel column chromatography on the eluent fractions (chloroform: methanol = 24: 1) gradient to chloroform / methanol = 100: 0 to 24: And fractionation of FE5 (chloroform: methanol = 97: 3) in chloroform / methanol = 100: 0 to 24: 1 among the eight fractions was carried out by silica gel column chromatography to obtain four fractions FE5-1 to FE5-4), and the fractions of FE5-1 and FE5-2 in the four fractions were combined, and water containing acetonitrile / 0.1% acetic acid = 5: 95-100: 0 And subjected to C18-reversed phase 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 above-mentioned fair-forbased compound and its tautomer can be prepared by the following steps:

1) adding soybean oil to ethyl acetate to obtain an extract of soybean oil ethyl acetate; And

2) Performing column chromatography using the chloroform / methanol as an elution solvent to extract the soybean leaf extract obtained in the above Step 1 to separate and purify the compound.

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

The step 1) is a step of extracting the soybean leaf with ethyl acetate.

In the above step, the method for obtaining the ethyl acetate extract is as follows. The dried soybean leaves are cut into an extraction container, and ethyl acetate is added thereto to extract the extract at room temperature. The above procedure may be repeated several times in order to increase the extraction efficiency of the bean leaves. Thereafter, the extract is allowed to stand for a predetermined time, and then filtered with a filter paper or the like to produce an ethyl acetate extract.

The step 2) is a step of separating and purifying the compound by performing column chromatography using an organic solvent as an elution solvent, the ethyl acetate extract obtained in the step 1).

This step can be carried out in the same manner as step 3) of the above-mentioned production method.

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

In addition, the present inventors conducted nuclear magnetic resonance (NMR) and mass spectrometry for the structural analysis of the separated compounds, and as a result, the compound was confirmed to be fairabide a represented by the formula (2) and its tautomer 1 to 11).

In addition, the present inventors have conducted measurement of the inhibitory activity of? -Glucosidase to verify the activity related to the metabolic diseases of the phaforobide compound represented by the formula (2) and its tautomer, and as a result, 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 [mu] M (51.0 [mu] g / ml), respectively.

In addition, the present inventors have conducted a toxicity test by orally administering the present compound of the present invention to a mouse. As a result, the above-mentioned fair-forbidden compound has an LD ₅₀ of at least 1000 mg / kg. < / RTI >

Therefore, the pharmaceutical composition of the present invention effectively inhibits? -Glucosidase activity and has little toxicity. Therefore, it can be effectively used as a pharmaceutical composition for the prevention and treatment of metabolic diseases including diabetes or its complications .

The present invention includes both a phaforobide compound represented by formula (2) and a tautomer thereof, as well as a pharmaceutically acceptable salt thereof, and a possible solvate, hydrate, racemate, or stereoisomer thereof which can be prepared therefrom do.

The phaforobide compound represented by the general formula (2) of the present invention and its tautomer can be used in the form of a pharmaceutically acceptable salt, and the salt includes an acid addition salt formed by a pharmaceutically acceptable free acid 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 can be prepared by a conventional method, for example, by dissolving at least one compound selected from the group consisting of a phospabide compound represented by the general formula (2) and a tautomer thereof in an excess amount of an acid aqueous solution, , For example, methanol, ethanol, acetone or acetonitrile.

It is also possible to prepare by the same amount of the phaforobide compound represented by the general formula (2) or a tautomer thereof, and an acid aqueous solution or alcohol, followed by evaporating the mixture and drying or filtrating the precipitated salt 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 the composition is formulated, it is prepared using diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, surfactants and the like which are usually used.

Solid preparations for oral administration include tablets, patients, powders, granules, capsules, troches and the like, which are prepared by mixing at least one of the fairaboidal compounds of formula (II) One or more excipients such as starch, calcium carbonate, sucrose or lactose, or gelatin. 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 body weight of the patient. In general, 0.1 mg to 100 mg, preferably 0.5 mg to 10 mg per kg of body weight is administered daily or every other day Or one to three 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.

The present invention also relates to a pharmaceutical composition for preventing or ameliorating metabolic diseases or complications thereof, comprising a pheophorbide-based compound represented by the following formula (1), a tautomer thereof or a pharmaceutically acceptable salt thereof as an active ingredient A food composition comprising:

[Chemical Formula 1]

.

.

Wherein R ¹ , R ² and R ³ are independently selected from H, C ₁ -C ₄ straight or branched alkyl, carboxyl, C ₁ -C ₄ alkoxycarbonyl, C ₁ -C ₄ alkoxycarbonyl C ₁ - C ₁ -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 linear or branched alkyl, C ₁ -C ₄ straight or branched alkoxy, amino and amino C ₁ -C ₄ alkyl It is either.

Preferably, the pearoboid-based compound is represented by the following formula (2), but is not limited thereto:

 (2)

.

.

In a specific example of the present invention, the present inventors conducted a measurement of the inhibitory activity of? -Glucosidase to verify the activity related to the metabolic diseases of the parenteral compound of formula (2) and its tautomer, 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) (45.2 μg / ml) and 86.1 μM (51.0 μg / ml), respectively (see Table 1).

In addition, the present inventors have conducted a toxicity test by orally administering the present compound of the present invention to a mouse. As a result, the above-mentioned fair-forbidden compound has an LD ₅₀ of at least 1000 mg / kg. < / RTI >

Therefore, the parenteral compound of the present invention effectively inhibits the activity of? -Glucosidase and has little toxicity. Therefore, it can be effectively used as a composition for a health food for preventing or ameliorating a metabolic disease including diabetes or its complications have.

There is no particular limitation on the kind of the food to which the pervapored compound of the present 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 phaforobide compound of the present invention can be added directly to food or used with other food or food ingredients, and can be suitably used according to conventional methods. 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 consumption intended for health or hygiene purposes or for health control purposes, 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. In addition, it may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks.

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

In addition,

1) fermenting the soybean leaves;

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

3) performing a column chromatography using an organic solvent as an extraction solvent to separate and purify the compound obtained in the step 2), and then mass-producing the peptide compound.

In a specific example of the present invention, the inventors of the present invention found that the 70% ethanol extract of the hot-air dried soybean leaves after aging according to the present invention was not aged, , The peak area of the pha promvide a was markedly increased compared with the 70% ethanol extract of the present invention, and this result was confirmed to be consistent with the increase in the inhibitory activity of? -Glucosidase activity.

Accordingly, it was confirmed that the content of the pea forbide compound according to the present invention increased through aging or fermentation using a microorganism of canola 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 Fairforbide system  Separation of compounds

<1-1> Preparation of soybean leaf extract

Glycine max soybean beans were sown in Jeungpyeong, South Chungcheong Province and harvested after 90 days of growth. 300 g of the dried bean curd was extracted with 3 L of ethyl acetate and extracted at room temperature for 3 days. The primary ethyl acetate soluble fraction was recovered using a filter paper. The residue of the bean curd leaf was extracted once again by the same method, The acetate-soluble portion was recovered. The primary and secondary ethyl acetate solubles were combined and concentrated under reduced pressure to give 13 g of ethyl acetate extract.

<1-2> Fairforbide system  Separation of compounds

The ethyl acetate extract (13 g) obtained in Example <1-1> was separated using a purification system (SP4 HPFC, biotage, Sweden, Uppsala). The silica gel column used was a mixture of chloroform and methanol as the eluting solvent under conditions of FLASH 75 + M (Biotage SNAP cartrige, 75 mm × 150 mm), flow rate of 75 ml / min and detection of the separated fraction of UV 254 nm The eluate was eluted with a gradient of chloroform / methanol = 100: 0 to 9: 1. One tube (75 ml) per tube was separated into a total of 100 tubes. Thin layer chromatography and UV absorbance The tubes containing the compounds with similar Rf values were combined and fractionated into 10 fractions (A to J).

The fractions E (chloroform: methanol = 24: 1, 2.8 g) were subjected to silica gel column chromatography with gradient of chloroform / methanol = 100: 0 to 24: 1 to obtain 8 fractions (FE1 to FE8) , And the fraction of FE5 (chloroform: methanol = 97: 3, 862 mg) was subjected to silica gel column chromatography with gradient of chloroform / methanol = 100: 0 to 24: ), Fractions of FE5-1 and FE5-2 (172 mg) were taken out of the four fractions, and water containing acetonitrile / 0.1% acetic acid was added to the mobile phase (water containing 0.1% acetic acid Between 20 and 35 min, 95 to 100% B for 35 to 40 min (solvent A) and acetonitrile (solvent B); a linear gradient elution program; Reverse phase high performance column chromatography (HPLC, RP-C18, Cosmosil 5C18-MS-II, 10 x 250 mm, 100% B for 40-45 min; 100-5% B for 45-55 min) 5 [mu] m, 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 bean leaves Fairforbide system  Structural analysis of compounds

NMR analysis and mass spectrometry were performed to analyze the structure of the compound isolated in Example 1 above.

<2-1> Proton nuclear magnetic resonance spectrum ( ^One H NMR spectrum ) Measurement and analysis of

As a result of the proton nuclear magnetic resonance spectrum ( ¹ H NMR spectrum) of the compound 1 isolated in Example 1, it was found that there were 6 aromatic or olefinic groups between 9.20 and 6.0 ppm, ) Proton signals were observed and four methyl peaks at 2.7 and 2.1 ppm and two methine proton at 4.48 and 4.11 ppm at 3.89, 3.53, 3.28 and 2.89 ppm, Two methyl peaks were observed at the unequal methylene peaks at 1.80 and 1.51 ppm (Fig. 1).

<2-2> Carbon Nuclear Magnetic Resonance Spectrum ( ¹³ C NMR spectrum ) Measurement and analysis of

For the structural analysis of the compound 1 isolated in Example 1, a carbon nuclear magnetic resonance spectrum ( ¹³ C NMR spectrum) was measured. As a result, it was found that a large number of carbonyl carbon, aromatic 23 due to the sp ² (aromatic), and the olefin group of carbon (carbon olefinic) 11 sp ³ carbon peaks attributed to carbon and methine, methylene and methyl carbon were confirmed (FIG. 2).

<2-3> Proton-Proton Two-dimensional Cozy Spectrum ^One H- ^One H COZY spectrum ) Measurement and analysis of

To confirm the partial structure of the compound 1 isolated in Example 1, a proton-proton two-dimensional COZY spectrum ( ¹ H- ¹ H COZY spectrum) was measured and analyzed (FIG. 3).

As a result, the presence of three partial structures, CH ₂ ═CH-, CH ₃ -CH-CH-CH ₂ -CH ₂ -, and CH ₃ -CH ₂ - was identified as shown in FIG. 6 (FIG. 6).

&Lt; 2-4 > Spectra of heteronuclear multiple quantum correlation spectroscopy ( HMQC spectrum ) And a heteronuclear multi-bond correlation spectroscopy spectrum ( HMBC spectrum ) Measurement and analysis of

In order to analyze the structure of the compound 1 isolated in Example 1, a proton-bearing carbon ( ¹ J _{C -H} ) was confirmed by measuring and analyzing the spectrum of the HMQC spectrum of the heteronuclear multiple quantum cascade (HMQC spectrum) 4).

From the long-range correlation observed in sp2 carbon from methyl proton at 3.53, 3.28, and 2.89 ppm, the methyl groups showed strong ring current effect (anisotropic effect ) (Fig. 5). This suggests that the present compounds may also be chlorophyll compounds due to the phenomena observed in chlorophyll compounds. Therefore, the cross-peaks in the HMBC spectrum were analyzed in comparison with the chlorophyll structure, and the results are shown in FIG. Based on this, the chemical structure of Compound 1 represented by the following Chemical Formula 2 was confirmed, and a database search was performed based on the chemical structure. As a result, it was confirmed that the present compound was pheophorbide a Respectively.

(2)

.

.

<2-5> Mass spectrum ( Mass spectrum ) Measurement and analysis of

From the nuclear magnetic resonance (NMR) spectroscopic analysis, the chemical structure of Compound 1 was identified as peophorbide a, and mass analysis was carried out for its confirmation.

Specifically, the ESI-mass was measured in a negative mode to confirm the molecular weight. That is, [MH] - was observed at m / z 591 and the molecular weight was found to be 592, which was confirmed to be exactly the same as the chemical structure of the NMR spectroscopic analysis (FIG. 8).

<2-6> Nuclear Magnetic Resonance Spectrum ( NMR spectrum ) Measurement and analysis of

For the analysis of the structure of Compound 2 isolated in Example 1, CDCl ₃ : CD ₃ OD in a mixed solvent to measure the proton nuclear magnetic resonance spectrum ( ¹ H NMR spectrum).

As a result, as shown in FIG. 11, Compound 2 was the same as Compound 1, but the 2.89 ppm methyl group migrated to the high magnetic field, and the 8.96 ppm methine proton migrated to the author's field It was confirmed that the present compound is a tautomer of a ternary complex produced by tautomerism of falphobide a (FIG. 11).

< Experimental Example  1> Fairforbide system   The α- Glucosides (? glucosidase ) activation Low performance  Measure

The pearoboid system isolated in Example 1 above The following experiment was carried out to investigate the effect of the compound on alpha -glucosidase activity essential for carbohydrate metabolism.

The inhibitory activity against? -glucosidase was partially modified by the nitrophenol method of Kato et al. (J. Med. Chem. 48: 2036-2044, 2005). p-nitrophenyl-D-glucopyranoside (Sigma-Aldrich) was hydrolyzed by? -glucosidase (EC 3.2.1.20, Baker Yeast) The accumulation amount of the generated chromophore was measured by absorbance.

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

A pair of compound α-glucosidase inhibitory activity (IC ₅₀ , μM) (2) 76.4 (or 45.2 μg / ml) The tautomer of formula (2) 86.1 (or 51.0 [mu] g / ml)

As shown in Table 1, the fair-forbased compound according to the present invention showed an IC ₅₀ value that was 50% inhibitory for the activity of? -Glucosidase, and in particular, Compound 1 and Compound 2 The IC ₅₀ values of the tautomers thereof were confirmed to be 76.4 μM (45.2 μg / ml) and 86.1 μM (51.0 μg / ml), respectively, which are shown to inhibit α-glucosidease activity (Table 1).

< Experimental Example  2> by aging or fermentation Fairforbide system  Increase of compound content

The following experiment was conducted to investigate the effect of the pearoboid-based compound isolated in Example 1 on the increase of the content and the activity according to the ripening of the bean leaf.

The harvested cane leaves were sealed in a bag and allowed to stand in the sun for 2 days before drying to obtain aged canola leaves. Generally, aging is a chemical process that slowly ferments or forms colloidal particles (particles) when a substance is left at a suitable temperature for a long time (time) In the present invention, soybean leaves are fermented using microorganisms (fungi etc.) polluted in a natural space. For artificial fermentation, it is not limited to contaminated microorganisms in a natural space, but can be used as an edible lactobacillus, yeast, Bacterium, Bacillus, and the like can be used. The bean leaves were ripened and then extracted in the same manner as in <Example 1>. The weight of the 70% ethanol extract, the 95% ethanol extract, or the ethylacetate extract obtained from the aged soybean leaves was 30.0 mg, 19.0 mg and 8.5 mg, respectively. Therefore, the weight of aged bean leaf extract was increased or decreased (Table 2).

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

Soybean extract Soybean leaf extract dry weight
(mg / 100 mg) ? -glucosidase inhibitory activity (%) Hot wind dried bean leaf 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 aging
Hot wind dried bean leaf 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 the above Table 2, when the hot-air dried soybean leaves after aging according to the present invention were compared with the non-aged hot-air dried soybean leaves, the content of α-glucoside increased from 70% ethanol extract, 95% ethanol extract or ethyl acetate extract Day activity inhibition.

To obtain the results, we analyzed the contents of each of the extracts by high performance liquid chromatography (HPLC) to determine the change in the content of the phaforobide compounds.

Specifically, a 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) , And Brownlee SPP C18-MS-II (4.6 x 50 mm, 2.7 μm) was used as the HPLC column. Acetonitrile / water containing 0.1% acetic acid was diluted with mobile phase {water (solvent A) containing 0.1% acetic acid 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 to 5% B for 45 to 55 min} at a flow rate of 1.8 ml / min. The results are shown in FIG. 12 to FIG.

As a result, as shown in FIG. 12 to FIG. 14, the pea of fairabide a was confirmed at 20.2 minutes, and the 70% ethanol extract of the hot-air dried bean leaves after aging according to the present invention showed 70 % Ethanol extract, the peak area of the pha promvide a was markedly increased, confirming that the result is in agreement with the increase in the inhibitory activity of? -Glucosidase activity.

Accordingly, it was confirmed that the content of the pea forbide compound according to the present invention increased through aging or fermentation using a microorganism of cannabis leaf (Figs. 12 to 14).

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

In order to examine the acute toxicity of the pearopoidal compounds contained in the composition according to the present invention, the following experiment was conducted.

<3-1> ICR  Manufacture of mice

Specific pathogens free ICR mice at 4 weeks of age were housed in an animal room of 12 L / 12D at a temperature of 22 ± 3 ° C, a humidity of 55 ± 10%, and 12 males and 12 males (3 females / Respectively. 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

(50 mg / ml) of 0.5% Tween 80 as a solvent, and then injected with 0.2 ml (500 mg / kg) per 20 g of mouse body weight, , And 0.4 ml (1,000 mg / kg), respectively.

Samples were administered orally once, and the following side effects or deaths were observed for 7 days after administration. That is, on the day of administration, changes in general symptoms and presence or absence of dead animals were observed at 1 hour, 4 hours, 8 hours, 12 hours after the administration, and 1 day at the morning and at the afternoon from the next day to 7 days after administration. Animals were sacrificed on the 7th day after administration, and the internal organs were examined visually. Changes in body weight were measured at intervals of one day from the day of administration to observe the animal weight loss phenomenon by the pearoboid compound.

As a result, no clinical symptoms were observed in all of the mice to which the test substance was administered, no mortal mice were observed, and no toxic changes were observed in weight changes, blood tests, blood biochemical tests, and autopsy findings. Therefore, based pair Four carbide represented by the formula (2) compound did not show any toxicity changes to 1000 mg / kg in all the mice, was determined by oral administration of a minimum lethal dose (LD ₅₀₎ is at least 1,000 mg / kg or more safe substance.

Accordingly, the parenteral compound of the present invention can be effectively used as a composition for preventing or treating metabolic diseases including diabetes mellitus or its complications, in which the side effects are alleviated because alpha-glucosidease activity is effectively inhibited.

< Manufacturing example  1> Manufacture of Pharmaceuticals

One. Sanje  Produce

500 ng of the &lt; RTI ID = 0.0 &gt;

Lactose 1 g

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

2. Preparation of tablets

500 ng of the &lt; RTI ID = 0.0 &gt;

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

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

3. Preparation of capsules

500 ng of the &lt; RTI ID = 0.0 &gt;

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

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 the &lt; RTI ID = 0.0 &gt;

180 mg mannitol

Na2HPO42H2O 26 mg

Distilled water 2974 mg

According to the conventional method for preparing an injectable preparation, an injectable preparation was prepared by incorporating the aforementioned components in the amounts indicated.

< Manufacturing example  2> Manufacture of health food

1. Manufacture of health food

500 ng of the &lt; RTI ID = 0.0 &gt;

Vitamin mixture quantity

70 [mu] g of vitamin A acetate

Vitamin E 1.0 mg

0.13 mg of vitamin

0.15 mg of vitamin B2

Vitamin B6 0.5 mg

0.2 [mu] g vitamin B12

Vitamin C 10 mg

Biotin 10 μg

Nicotinic acid amide 1.7 mg

Folic acid 50 mg

Calcium pantothenate 0.5 mg

Mineral mixture quantity

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Secondary calcium 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 the &lt; RTI ID = 0.0 &gt;

Citric acid 1000 mg

100 g of oligosaccharide

Plum concentrate 2 g

Taurine 1 g

Purified water was added and the entire 900 ml

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

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

Claims (8)

1) sealing the soybean leaves and leaving them in the sun to prepare aged soybean leaves;
2) adding 70% ethanol, 95% ethanol or ethyl acetate to the aged soybean leaf of step 1) to obtain a soybean leaf extract; And
3) column chromatography using the chloroform-methanol mixture as an eluting solvent, extracting and purifying the compound obtained in step 2);
&Lt; / RTI &gt; and a tautomer thereof.
delete delete delete 2. The method according to claim 1, wherein the pair of aphaabide a and a tautomer thereof exhibit an inhibitory effect of? -Glucosidase.
delete delete delete

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