KR101392478B1 - Composition Comprising Octaphlorethol A for Lowering Blood Glucose - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for lowering blood glucose, comprising an octafluorosulfur compound represented by the following formula as an active ingredient. More specifically, the present invention relates to a composition for lowering blood glucose, comprising octafluorosulfur as an active ingredient, A composition for preventing or treating diabetes or diabetic complications, and a health functional food showing prevention and improvement effects of diabetes or diabetic complications. The octafluoroserine compound according to the present invention has an effect of reducing blood sugar when administered to an animal model in which diabetes has been induced and is excellent in the inhibitory activity of the carbohydrate digestive enzymes alpha-glucosidease and alpha-amylase, It can be used for pharmaceutical use that can prevent and treat diabetes or diabetic complication induced by hyperglycemia and food use which can improve diabetes or diabetic complication.
≪

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for reducing blood glucose content, which comprises an octafluorosulfuron compound as an active ingredient. [0002] Composition Comprising Octaphlorethol for Lowering Blood Glucose [

The present invention relates to a novel compound which is separated and identified from broad-leaved brown algae among brown algae, and a process for producing the same.

The death rate due to diabetes in the Korean population increased from 11.8 per 100,000 population in 1990 to 22.6 in 2000, increasing by 91.5% over the last 10 years. The number of deaths increased over time, Diabetes mellitus is the fourth largest (Korea National Statistical Office. Annual Report on the Cause of Death Statistics 19. 1999).

In general, diabetes mellitus is defined as a series of metabolic diseases characterized by hyperglycemia and is known to be caused by an abnormally high plasma glucose level in humans and mammals. In addition, these abnormal glucose levels increase the level of hemoglobin in the plasma, and may be associated with chronic hyperglycemia, atherosclerosis, microangiopathy, kidney disease, heart disease, diabetic retinopathy diabetic retinopathy, and cataracts.

Insulin dependent diabetes (IDDM), which is an insulin-dependent type, is a type of insulin-dependent diabetes mellitus, which is caused by the secretion of insulin (glucose-regulating hormone in the blood) It is also called juvenile diabetes because it occurs mainly in young people aged 10 to 20 years. Type II diabetes (non-insulin dependent diabetes, NIDDM) occurs mainly after the age of 40, and it accounts for most of the diabetic patients in Korea. Unlike type Ⅰ, it is called adult type diabetes. The cause of the disease is not known yet, but it is known that genetic factors and environmental factors are involved together. As a pathogenesis of type II diabetes, both insulin secretion defects in pancreatic beta cells and insulin action defects in target cells (insulin resistance) are observed, although it is not yet clear which change is of primary importance.

Currently, there are five classes of compounds used for complications such as type 2 diabetes and insulin resistance: biguanides, thiazolidinediones, sulfonylureas, benzoic acid, Derivative compounds and? -Glucosidase inhibitors have been used. Among these, it has been known that acetylated compounds such as metformin interfere with excessive gluconeogenesis in the blood. Thiazolidinedione compounds are believed to act to increase the glucose consumption rate of the peripheral nerves and are believed to be useful in the treatment of diabetic complications such as sulfonylurea, such as tolbutamide and glyburide, benzoic acid, such as repaglinide, Derivatives and? -Glucosidase inhibitors such as acarbose act to lower plasma glucose by stimulating insulin secretion.

However, sulfonylurea compounds can not be administered to insulin-dependent type I diabetic patients. In the case of non-insulin dependent type Ⅱ diabetes mellitus, insulin secretion is reduced. In women, abnormal fetal birth, abortion, (stillbirth). In addition, most sulfonylurea compounds should be administered with caution to patients with impaired hepatic and renal function due to the sulfonylurea metabolism.

Although the mechanism of the acetylated formulations such as metformin has not been well established, the acetylated compounds are more effective than the sulfonylureas in lowering the insulin secretion of the pancreas, low. However, it may cause nausea, vomiting, diarrhea, and rash at the beginning of the administration, and may cause side effects such as fatal lactic acidosis. Therefore, it can be used only as a clinical test reagent in the United States.

Therefore, sulfonylurea and biguanide-containing preparations have such drawbacks and side effects, and therefore, there is a need for an excellent hypoglycemic agent having low side effects and high safety. In addition, the cure of diabetes mellitus has not been established so far, and there are many limitations on drug use due to the side effects of drug treatment. Therefore, studies to search for diabetes drugs from natural products have been actively conducted, and WHO (1990) (Grover JK, Vats, V., Rathi, SS, Journal of Ethnopharmacology, 73, pp461-470, 2000). Therefore, it is urgent to find new pharmacological substances derived from natural materials as a substance capable of lowering blood sugar.

On the other hand, among the seaweeds, the larvae belonging to the brown algae give a short circumferential stem at the basal end of a small anaglyph, and the upper part has a broad idea or object, and branches off. The tip is round, and sometimes there is air in the tissue at the end of the branch, so it may expand like bubbles. It grows from spring to summer and is known to be distributed in China, Japan, and Mexico inhabiting the southern coast, west coast, and Jeju island coast.

 Studies on various physiological activities possessed by broad-spectrum have also been continuing. According to the results of previous studies, Korean Patent No. 0745021 discloses that the extract of Wisteria sp. Has antimicrobial activity against causative agents of acne and thus can be used as a cosmetic composition for acne skin Korean Patent Laid-Open Publication No. 2003-0015536 discloses that whiting extract has whitening activity, and Korean Patent No. 0386417 discloses that whiting has a strong antifouling activity against marine adherent organisms, It is disclosed that it can be used for the development of an antifouling agent.

However, there has been no study on the hypoglycemic effect of such hypertrophy.

Accordingly, the present inventors have completed the present invention by confirming that octafluoroserine compound having an antidiabetic activity is separated from broad-leaved brown algae among seaweeds and that the compound has an action to improve antidiabetic activity and insulin resistance Respectively.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for lowering blood glucose containing an octafluoroserine compound as an active ingredient.

Another object of the present invention is to provide a health functional food for preventing or ameliorating diabetes comprising the octafluoroserine compound as an active ingredient according to the present invention.

In order to achieve the above object, the present invention provides a pharmaceutical composition for reducing blood glucose content, which comprises an octafluoroserine compound as an active ingredient.

In one embodiment of the invention, the compound may be derived from Ishige foliacea.

In one embodiment of the present invention, the octafluoroserine compound may be prepared by adding methanol to a wister powder to obtain a widespread methanol extract; Adding water and ethyl acetate to the obtained extract to obtain an ethyl acetate fraction; Ethyl acetate fraction is carried out by performing silica gel chromatography using chloroform and a methanol solvent; The fraction obtained through silica gel chromatography was subjected to Sephatex LH-20 column chromatography using a methanol solvent; And fractions obtained through Sephatex LH-20 column chromatography can be obtained again by carrying out high performance liquid chromatography (HLPC).

In one embodiment of the present invention, the chloroform and the methanol solvent may be mixed in a ratio of 50: 1 to 1: 1 in performing the silica gel chromatography using the chloroform and the methanol solvent.

In one embodiment of the present invention, 100% methanol was used as the eluent solvent used in the Sephatex LH-20 column chromatography, and methanol as the elution solvent used in the high performance liquid chromatography (HLPC) To 40% methanol.

In one embodiment of the present invention, the octafluoroserine compound may have a digestive enzyme inhibitory action, a glucose absorption promotion action in muscle, and an insulin resistance improving action of carbohydrate.

The present invention also provides a health functional food for preventing or ameliorating diabetes comprising the octafluoroserine compound according to the present invention as an active ingredient.

The present invention relates to a composition for reducing blood sugar containing octafluoroserine as an active ingredient, a composition for preventing or treating diabetic or diabetic complications, and a health functional food exhibiting an effect of preventing or improving diabetic or diabetic complications. The octafluoroserine compound according to the present invention has an effect of reducing blood sugar when administered to an animal model in which diabetes has been induced and is excellent in the inhibitory activity of the carbohydrate digestive enzymes alpha-glucosidease and alpha-amylase, It can be used for pharmaceutical use that can prevent and treat diabetes or diabetic complication induced by hyperglycemia and food use which can improve diabetes or diabetic complication.

FIG. 1 is a schematic diagram illustrating a process of separating an octafluoroserine compound having a hypoglycemic activity from a wort in an embodiment of the present invention.
2 is a graph showing the inhibitory activity of alpha-glucosidase of an octafluoroserine compound.
3 is a graph showing the inhibitory activity of alpha-amylase of octafluoroserine compound.
FIG. 4 is a graph showing blood glucose lowering effect of octafluoroserine compound in streptozotocin (STZ) -induced diabetic rats.
FIG. 5 is a graph showing the blood glucose lowering ability of octafluorosine compound in normal rats.
6 is a graph showing the degree of glucose uptake into muscle cells of the octafluoroserine compound.
FIG. 7 shows the result of western blotting of the effect of the octafluoroserine compound on the glucose uptake into muscle cells.
8 is a graph showing blood glucose lowering effects of octafluoroserine compound in a rat of type 2 diabetes mellitus.

The present invention is characterized by providing a pharmaceutical composition for lowering blood glucose containing an octafluoroserine compound as an active ingredient.

Specifically, the octafluoroserine compound according to the present invention is a compound isolated from a skimmed liquor, which is 2- (4- (4- (4- (4- (4- (4- (3,5- dihydroxyphenoxy) -3,5-dihydroxyphenoxy) -3,5-dihydroxyphenoxy) -3,5-dihydroxyphenoxy) -2,6-dihydroxyphenoxy) -2,6-dihydroxyphenoxy) -2,6- 5-triol. ≪ / RTI >

≪

The octafluoroserine compound separated from the wool according to the present invention may be isolated from nature or may be prepared by a chemical synthesis method well known in the art. Preferably, methanol is added to the wool powder, ; Adding water and ethyl acetate to the obtained extract to obtain an ethyl acetate fraction; Ethyl acetate fraction is carried out by performing silica gel chromatography using chloroform and a methanol solvent; The fraction obtained through silica gel chromatography was subjected to Sephatex LH-20 column chromatography using a methanol solvent; And fractions obtained through Sephatex LH-20 column chromatography can be obtained again by carrying out high performance liquid chromatography (HLPC).

More specifically, the method of separating and purifying the compound of the present invention from a broad bean can be described as follows. First, the bean curd sold in the market can be used first. In one embodiment of the present invention, We used a clean cloth that was cleaned several times.

The resultant wort can be first pulverized, wherein the wort can be pulverized using a pulverizer, preferably pulverized to a size of 50 mesh or less.

When the pulverization step of the wort is completed, methanol is added to the wort powder, and the wort powder is immersed in methanol to obtain a methanol extract. The amount of methanol to be added at this time is 10 to 15 times the weight of the wort powder can do.

In addition, the solvent extraction step of extracting with methanol may be carried out under the conditions of temperature and time generally used in the art. Preferably, methanol is added to the wollastonite powder and the mixture is stirred at 20 to 40 ° C for 20 to 28 Extract can be obtained by extracting for a time.

In this case, when the extraction is carried out at a temperature of less than 20 ° C for less than 20 hours, there is a problem that the octafluorosulfuric acid compound contained in the wort can not be extracted in a sufficient yield, When the extraction is carried out at a time exceeding the time, there may arise a problem that the activity of the octafluoroserine compound is decreased due to the high temperature and long time extraction reaction. Therefore, it is preferable to carry out the reaction under the extraction conditions.

In addition, when the extraction process is completed, centrifugation can be performed for 10 to 20 minutes to remove the unextracted residue. Further, in order to obtain as much compounds as possible from the loose leaf, the residue collected through centrifugation The extraction of the above method using an organic solvent can be repeated two or three times in the same manner.

When the extract is obtained using methanol, the resulting extract is concentrated, the concentrate is dissolved in water, and ethyl acetate is added thereto to obtain an ethyl acetate fraction.

The obtained ethyl acetate fraction can then be subjected to various column chromatography to obtain a fraction containing the octafluorosine compound, and the purification process can be preferably performed by performing silica gel chromatography.

In this case, the silica gel chromatography may use chloroform and a methanol solvent as a separation solvent (elution solvent), preferably, an ethyl acetate fraction is injected into silica gel chromatography, and a chloroform: methanol solvent is mixed at a mixing ratio of 50: 1 to 1: Can be carried out by mixing.

The fraction thus obtained through silica gel chromatography can be further subjected to Sephadex LH-20 column chromatography, which can be carried out using 100% methanol as a separation solvent.

The fraction obtained by performing Sephadex LH-20 column chromatography can be further purified by goethanol liquid chromatography again, using 30-40% methanol as a separation solvent.

The compound obtained by carrying out such a purification process can be confirmed by mass analysis (for example, using ESI-Mass analysis) and nuclear magnetic resonance analysis (NMR) of the substance structure and chemical formula identification. The isolated compound identified above has the formula: " 2- (4- (4- (4- (4- (4- (3,5-dihydroxyphenoxy) -3 Dihydroxyphenoxy) -3,5-dihydroxyphenoxy) -2,6-dihydroxyphenoxy) -2,6-dihydroxyphenoxy) -2,6-dihydroxyphenoxy) benzene- 1,3,5- And it was named Octafloresol.

Furthermore, the octafluorosulfan compound purified by the method of the present invention has an activity of lowering blood glucose, and thus is capable of preventing and treating diabetes.

That is, according to one embodiment of the present invention, the effect of the octafluoroserine compound on the activity of the carbohydrate digesting enzymes, alpha-glucosidase and alpha-amylase, , It was shown that the inhibitory activity of alpha-glucosidase and alpha-amylase was increased in proportion to the concentration of the treated octafluoroserine compound, and this inhibitory activity was inhibited by the conventional alpha-glucosidase and alpha-amylase inhibitory activity Similar to or better than acarbose known to have (see Figures 2 and 3).

In general, carbohydrate digestion enzymes are enzymes that break down carbohydrates contained in diets into polysaccharides and disaccharides and monosaccharides. Inhibitors that inhibit carbohydrate digestion enzymes can delay the digestion and absorption of carbohydrates, thereby alleviating postprandial hyperglycemia.

Therefore, in the case of the carbohydrate digestive enzyme inhibitor, the octafluoroserine compound of the present invention is used for the prophylaxis and treatment of diabetes mellitus, because it is used as a therapeutic agent for a disease caused by an increase in blood glucose such as diabetes through alleviating the increase in blood glucose It can be used.

Further, according to another embodiment of the present invention, the present inventors have confirmed this fact through an experiment on animal models. That is, according to another embodiment of the present invention, when an octafluorosine compound is treated on diabetic mice, (Fig. 4 and Fig. 5).

On the other hand, as described in the prior art, diabetes mellitus is generally defined as a series of metabolic diseases characterized by hyperglycemia. Such diabetes mellitus is classified as type 1 diabetes and type 2 diabetes Insulin dependent diabetes (IDDM) is caused by insulin secretion deficiency in the blood, and non-insulin dependent diabetes (NIDDM) is caused by insulin-dependent diabetes mellitus Is a type of diabetes mellitus that is known to be caused by insulin secretion in the pancreatic beta cells and defective insulin action in the target cells (insulin resistance).

In particular, type 2 diabetes reduces insulin action in peripheral tissues such as liver, muscle, and adipocytes, and decreases glucose utilization. In particular, when glucose is elevated in the liver, glucose levels increase in the pancreatic beta cells It is a disease that occurs when the insulin secretion is not enough to inhibit the rise. Type 2 diabetes mellitus is caused by the inability of insulin secretion to compromise insulin resistance. In addition, insulin is secreted in type 2 diabetes, but insulin action is reduced in the peripheral tissues such as liver, muscle, and adipose tissue where insulin acts. As a result, glucose utilization is decreased, It is accompanied by hyperinsulinemia, which is continuously secreted by insulin.

In addition, recently published data show that in the development of diabetes treatment, we are interested in the muscles that make up 45% of our body. The development of drugs that lower the blood sugar by increasing the reactivity to insulin in muscles is a good treatment for diabetes Unlike fats or hepatocytes, which are targeted and increased glucose uptake by insulin alone, glucose can be introduced into the muscle by insulin dependent and independent pathways. Therefore, it has been reported that a substance that accelerates absorption of glucose in muscles can be used as a therapeutic agent for preventing diabetes.

Accordingly, the present inventors have found that octafluorosole compound acts to absorb glucose into muscle cells. As a result, octafluorosole compound has an activity of increasing the expression of Akt, AMPK and GLUT4 protein, which are necessary proteins for glucose uptake into muscles And therefore, it was found that octafluoroserine absorbs glucose into muscles through the action of GLUT4, a glucose transporter, which moves to the cell membrane and absorbs glucose into the cells, due to the activity of Akt and AMPK ( 7).

In addition, according to another embodiment, octafluoroserine exhibited an activity of inhibiting blood glucose increase in an animal model of type 2 diabetes and exhibited activity of reducing insulin content in blood (see FIGS. 8 and 9) ).

Thus, the present inventors have found that the octafluoroserine compound can prevent and treat impaired glucose tolerance and hyperinsulinemia due to insulin resistance observed in the early stage of diabetes, and can prevent lipid metabolism disorders due to diabetes and hyperinsulinemia, diseases such as hypertension and coronary artery disease And thus can be used as a therapeutic agent.

Therefore, the present invention provides a pharmaceutical composition for hypoglycemia containing an octafluoroserine compound as an active ingredient.

The composition according to the present invention may contain an octafluoroserine compound in a pharmaceutically effective amount, wherein the pharmaceutically effective amount refers to an amount of the octafluorosine compound to prevent or reduce the symptoms of a disease requiring blood glucose lowering, And an amount sufficient to treat.

The pharmacologically effective amount of octafluoroserine according to the present invention is 0.5 to 100 mg / day / kg body weight, preferably 0.5 to 5 mg / day / kg body weight. However, the pharmaceutically effective amount may be appropriately changed depending on the degree of diabetic or diabetic complication symptoms, the age, body weight, health condition, sex, administration route and treatment period of the patient.

The term "pharmaceutically acceptable" as used herein refers to a composition that is physiologically acceptable and does not normally cause an allergic reaction such as gastrointestinal disorder, dizziness, or the like when administered to humans. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Further, it may further include a filler, an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and an antiseptic agent.

In addition, the compositions of the present invention may be formulated using methods known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to the mammal. The formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatine capsules, sterile injectable solutions, sterile powders.

The composition according to the present invention may be administered through various routes including oral, transdermal, subcutaneous, intravenous or muscular, and the dose of the active ingredient may be varied depending on various factors such as route of administration, age, sex, And the like.

In addition, the composition according to the present invention may be administered in combination with a known compound having an effect of preventing, ameliorating or treating the symptoms of diabetes or diabetic complications.

Accordingly, the present invention can provide a medicament which can prevent or treat the symptoms of diabetes or diabetic complications including a composition containing octafluoroserine or a pharmaceutically acceptable salt thereof as an active ingredient.

Furthermore, as described above, the composition of the present invention can be used not only as a pharmaceutical composition for the purpose of preventing or ameliorating the symptoms of diabetes or diabetic complications, but also for the prevention of diabetic or diabetic complications, It can be used as a food composition for manufacturing.

Therefore, the food composition of the present invention can be easily utilized as a food which is effective for prevention and improvement of diabetic or diabetic complication symptoms, for example, as a raw material, additives, food additives, functional foods or beverages of foods.

As used herein, the term " food " means a natural product or a processed product containing one or more nutrients, preferably a state capable of being directly eaten through a certain degree of processing, , Food, food additives, functional foods and beverages.

Examples of foods to which the composition for preventing or ameliorating the symptoms of diabetes or diabetic complication according to the present invention can be added include various foods, beverages, gums, tea, vitamin complex, and functional foods. In addition, in the present invention, the food may include special nutritive foods (e.g., crude oil, spirits, baby food, etc.), meat products, fish meat products, tofu, mackerel, noodles (Such as soy sauce, soybean paste, kochujang, mixed potatoes), sauces, confectionery (eg, snacks), candies, chocolate, gums, ice cream, milk products (eg, fermented milk, cheese, But are not limited to, pickled foods (various kinds of kimchi, pickles, etc.), beverages (e.g., fruit drinks, vegetable beverages, beverages, fermented beverages and the like) and natural seasonings (e.g. The food, beverage or food additive may be prepared by a conventional production method.

The above-mentioned " functional food " refers to a food group which is imparted with added value to function and express the function of the food by physical, biochemical or biotechnological techniques, or to control the bio-defense rhythm of the food composition, Refers to a food prepared by processing a body so as to sufficiently express the body's control function on the body, such as recovery, and the like. Specifically, it may be a health functional food. The functional food may include a food-acceptable food-aid additive, and may further comprise suitable carriers, excipients and diluents conventionally used in the production of functional foods.

In the present invention, the term " beverage " means a general term for drinking or enjoying a taste, and includes a functional beverage. The beverage includes a composition for preventing or ameliorating the symptoms of diabetes or diabetic complication as an essential ingredient at the indicated ratio. There are no particular restrictions on other ingredients, and various flavors or natural carbohydrates, such as ordinary beverages, .

In addition to the above-mentioned items, the food containing the composition for preventing or ameliorating the symptoms of diabetes or diabetic complication of the present invention may contain flavors such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, And carbonating agents used in fillers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, And these components can be used independently or in combination.

Furthermore, the present invention can provide a health functional food comprising octafluoroserine and a pharmaceutically acceptable food supplementary additive exhibiting the effect of preventing or ameliorating diabetes or diabetic complications.

In the food containing the composition for preventing or ameliorating diabetic or diabetic complication symptoms according to the present invention, the amount of the composition according to the present invention may be 0.001% by weight to 90% by weight, preferably 0.1 And may be contained in an amount of 0.001 g to 2 g, preferably 0.01 g to 0.1 g, based on 100 ml of beverage. However, for the purpose of health and hygiene, , The active ingredient may be used in an amount of more than the above range because there is no problem in terms of safety. Therefore, the scope of the present invention is not limited to the above range.

Hereinafter, the present invention will be described in detail with reference to examples. However, these examples are intended to further illustrate the present invention, and the scope of the present invention is not limited to these examples.

< Example  1>

From wide  Having a hypoglycemic activity Octafloresol  Isolation of Compound A

<1-1> Spread  Preparation of methanol extract

The seaweeds collected directly from the coast of Jeju were selected and washed with water several times, lyophilized, and pulverized using a grinder with a size of 50 mesh or less. Then, 5 L of 80% methanol was added to 50 g of pulverized wisteria, and the wister powder was immersed in the solution. The solution was subjected to solvent extraction at 20 ° C for 24 hours, and then centrifuged at low temperature to remove the residue. 4). The filtered methanol extract was concentrated by using a vacuum concentrator to obtain a methanol extract of Sprout.

<1-2> Spread  From methanol extracts Octafloresol Ai  Separation and purification

The 80% methanol extract (10 g) obtained above was dissolved in water (1 L), and silica gel having a diameter of 100 탆 was filled in a 55 x 400 mm glass tube. Ethyl acetate fractions were adsorbed on the silica gel, and chloroform: 1 to 1: 1 ratio. The separated active ingredients were loaded onto a column packed with a Sephadex LH-20 filler having a diameter of 100 mu m in a glass tube of 25 x 300 mm, and then eluted with 100% methanol to separate the active ingredient. Subsequently, the active ingredient was injected into high performance liquid chromatography to separate substances having excellent carbohydrate inhibitory activity and promoting glucose uptake in muscles.

The mass of the active ingredient was analyzed by ESI-mass (flow rate = 0.2 ml / min, 2.1 × 100 mm C-18 column; Thermo (USA), Thermo HPLC-mass system) Was measured by ¹ H-NMR (400 MHz, DMSO- d ₆ ) and ¹³ C-NMR (100 MHz, DMSO- d ₆ ).

As a result, it was confirmed that the compound identified through the method of the present invention was a compound having the following formula, and the molecular weight was found to be 994 MW by ESI-mass analysis. The formula was also found to be C ₄₈ H ₃₄ O ₂₄ and its chemical name was 2- (4- (4- (4- (4- (4- (3,5-dihydroxyphenoxy) dihydroxyphenoxy) -3,5-dihydroxyphenoxy) -3,5-dihydroxyphenoxy) -2,6-dihydroxyphenoxy) -2,6-dihydroxyphenoxy) -2,6-dihydroxyphenoxy) benzene-1,3,5- And named it "octafloresolei". The process of isolating and identifying the compound is shown in Fig.

&Lt; Structural formula of octafluorosine compound >

Further, it was found that the compound identified in the present invention had 24 double bonds and 17 hydroxyl groups through the molecular formula, and it was found that the compound had 8 ring structures.

Further, analysis by ¹ H-NMR (400 MHz, DMSO- d ₆ )

^{δ 1 H (mult, J =} Hz) is 5.57 (1H, d, J = 2.8 Hz, H-4), 5.58 (1H, d, J = 2.8 Hz, H-6), 5.59 (1H, d, J = 2.8 Hz, H-9) , 5.59 (1H, d, J = 2.8 Hz, H-11), 5.68 (1H, d, J = 1.8 Hz, H-15), 5.68 (1H, d, J = 1.8 Hz, H-17), 5.71 (1H, d, J = 1.8 Hz, H-21), 5.72 (1H, d, J = 1.8 Hz, H-23), 5.85 (1H, d, J = 1.6 Hz, H-26), 5.84 (1H , d, J = 1.8 Hz, H-30), 5.94 (1H, d, J = 1.8 Hz, H-32), 5.94 (1H, d, J = 1.8 Hz, H- 36), 6.15 (1H, d , J = 1.8 Hz, H-38), H-44 6.15 (1H, d, J = 1.8 Hz, H-42), 6.16 (1H, d, J = 1.6 Hz,) , 6.01 (1H, d, J = 1.6 Hz, H-46), and 6.16 (1H, d, J = 1.6 Hz, H-48), 9.02 (s, OH-1,3), 9.04 (s, OH OH, 8,12), 8.92 (s, OH-20,24), 9.06 (s, OH, -39, 41) and 9.07 (s, OH-45, 47)

Analysis using ¹³ C-NMR (100 MHz, DMSO- d ₆ )

? ¹³ C is? 153.0 (s, C-1), 153.0 (s, C-3), 156.1 (s, C-5), 152.9 151.1 (s, C-12), 151.1 (s, C-14), 154.1 (s, C-16) C-29), 154.0 (s, C-25), 154.1 (s, C-22), 151.1 (s, C-33), 150.8 (s, C-35), 156.2 (s, C-37), 152.7 C-2), 122.0 (s, C-7), 122.0 (s, C-9), 122.0 C-14), 123.4 (s, C-19), 123.4 (s, C-28), 123.5 ), 94.7 (d, C-4), 94.7 (d, C-11), 94.7 , 94.7 (d, C-26), 94.7 (d, C-30), 94.1 , 94.1 (d, C-42), 94.7 (d, C-44), 94.0 (d, C-46) and 94.7 (d, C-47).

< Example  2>

Octafloresol  The alpha-compound of the A- Glucosidase  Measurement of inhibitory activity

The inhibitory activity of alpha-glucosidase, which is a carbohydrate digestive enzyme of octafluoroserine, was measured as follows. First, 2 g / L bovine serum albumin (BSA) and 0.2 g / L sodium azide (NaN3) were dissolved in 100 mL of 100 mM PBS, and then 0.008 g of yeast α-glucosidase (Sigma) was dissolved to prepare an enzyme solution And a substrate solution was prepared by dissolving p-nitrophenyl-α-D-glucopyranoside in PBS at a concentration of 5 mM. 10 μl of a sample (Octaflores A) was added to 50 μl of the enzyme solution, and the absorbance before the reaction was measured at 405 nm, and then left at room temperature for 5 minutes. Then, 50 μl of the substrate solution was added, reacted for 5 minutes, and the absorbance was again measured to calculate the enzyme inhibitory activity from the change in absorbance. The inhibition rate of alpha-glucosidase in the calculated octafluoroserine is shown in Fig.

As a result, as shown in FIG. 2, it was confirmed that the octafluoroserine compound isolated in the present invention inhibited the activity of alpha-glucosidase, which is a carbohydrate digestive enzyme, in a concentration-dependent manner. It was found that it exhibited better inhibitory activity than acarbose, which is a commercially available alpha glucosidase inhibitor. In addition, Table 1 below shows the concentrations of octafluoroserine and acarbose inhibiting alpha-glucosidase by 50%, indicating that octafloresone inhibits alpha glucosidase by 50% at a much lower concentration than acarbose .

Accordingly, the inventors of the present invention have found that the octafluorosule identified in the present invention has a better activity of inhibiting alpha glucosidease than the conventional alpha glucosidees inhibitor acarbose, It can be used for the purpose.

Concentration of octafluoroserine and acarbose inhibiting alpha glucosidase by 50% Treated sample 50% inhibition of α-glucosidase (mg / mL) Acarbose 0.19 + 0.03 Octafloresole 0.11 ± 0.05 ^*

< Example  3>

Octafloresol  Measurement of alpha-amylase inhibitory activity of the A-compound

The inhibitory activity of alpha-amylase, the carbohydrate digesting enzyme of octafluoroserine, was measured by the following method. First, an enzyme solution was prepared by dissolving 0.1 g / L BSA, 0.01 g / L sodium azide (NaN3) and 0.2857 g porcine pancreas α-amylase (Sigma) in 50 mL of PBS to obtain an enzyme solution, and p- nitrophenyl-α-D-maltopentoglycoside (Sigma) was dissolved in PBS at a concentration of 5 mM to prepare a substrate solution. Then, 10 μl of each sample was added to 50 μl of the enzyme solution (octafluorosine was added at a concentration of 0.0625, 0.125, 0.25, 0.5 mg / ml), and the absorbance before the reaction was measured at 405 nm, 50 μl of the solution was reacted for 5 minutes, and the absorbance was again measured to calculate the enzyme inhibitory activity from the change in absorbance. The calculated inhibition rate of α-amylase of octafloresol A is shown in FIG.

As a result, it was shown that octafluoroserine inhibits the activity of alpha-amylase, a carbohydrate digestive enzyme, in a concentration-dependent manner. Acarbose, which is known as alpha amylase inhibitor, As shown in Fig.

Also, Table 2 below shows the concentrations of octafluoroserine and acarbose inhibiting alpha-amylase by 50%, indicating that octafluorosule inhibits alpha amylase by 50% at lower concentrations than acarbose. Therefore, the present inventors have found that the octafluoroserine compound has a better alpha-amylase inhibitory activity than the acarbose used for inhibiting the activity of the conventional alpha-amylase.

Comparison of the concentration of octafluoroserine and acarbose inhibiting 50% of alpha amylase Treated sample 50% inhibition of α-glucosidase (mg / mL) Acarbose 0.47 + 0.07 Octafloresole 0.34 + 0.06 ^*

< Example  4>

Octafloresol  Measurement of blood glucose lowering effect

Four - week - old male ICR mice were purchased from a central experimental animal and used for the experiment after 2 weeks' normal diet. The experimental group was randomly assigned to the normal group and the STZ - treated diabetic group. The temperature and humidity of the breeding room were maintained at 20 ± 2 ° C and 50 ± 10%, and the darkness and the humidity were adjusted at intervals of 12 hours. STZ administration Diabetic rats were injected intraperitoneally with streptozotocin (STZ, 150 mg / kg of body weight) dissolved in 0.1 M citrate buffer (pH 4.5) for 12 h fasting ICR mice. STZ administration The blood was taken from the tail vein of ICR mice in the fasting state and was used for the experiment when the blood glucose level measured by the hepatic glucose meter was more than 300 mg / dL. STZ - induced diabetic group and normal group were divided into control group, Octaflone group and acarbose group. ICR mice were fasted for 12 hours at night and blood was collected from the tail vein. Control group was treated with soluble starch (2g / kg of body weight) in ICR mice, group treated with octafluorosaldehyde and group treated with acarbose as a positive control group. The experimental group and the positive control group Oral administration of ICR mice in which starch (100 mg / kg body weight) and acarbose (100 mg / kg body weight) were dissolved in distilled water and starved for 12 hours was added to soluble starch (2 g / Respectively. Blood was collected from the tail vein at a fixed time (0, 30, 60, 120 minutes) after the administration.

As a result, as shown in FIG. 4, the group treated with the octafluoroserine compound of the present invention showed a decrease in blood glucose level in an animal model in which diabetes was induced, and in particular, the administration of starch 60 Minute, the blood glucose level was maintained lower than that of the control group. In other words, when the starch was administered to the STZ-induced diabetic rats, the blood glucose level was significantly increased in the control group, whereas the octafluoroserine group showed a lower blood glucose level than the control group, The blood glucose lowering effect similar to the postprandial hypoglycemic agent acarbose was shown.

FIG. 5 is a graph showing the hypoglycemic effect of octafloresolone on the normal rats. In the normal rats, the octafloxolane-treated group showed lower blood glucose level than acarbose, as compared with the control group. In other words, blood glucose level was significantly increased at 30, 60, 90, and 120 minutes after starch administration to the fasted control group, whereas starch and octafolestone were significantly increased compared to the control group, Increase in the number of patients. In addition, it was also confirmed that octafluorosulfate improves postprandial blood glucose increase similar to that of acarbose, which is used as a postprandial hypoglycemic agent.

< Example  5>

Octafloresol  Of this compound Into muscle cells  Measurement of effect on glucose uptake

The following experiment was carried out in order to examine the effect of the present octafluorodibenzoate on glucose uptake into muscle cells.

<5-1> Cell culture and differentiation

The muscle cells (L6) used in the experiment were purchased from Korean Cell Line Bank and cultured in DMEM medium supplemented with 10% FBS and 1% antibiotics at 37 ° C in a 5% CO ₂ incubator. If the undifferentiated muscle cells (L6) were 70 to 80%, DMEM medium supplemented with 2% horse serum was added and the differentiation was induced for 7 days and the medium was changed every 2 days.

<5-2> Into muscle cells  Glucose absorption measurement

The differentiated muscle cells were starvated in serum-free DMEM medium for 4 hours and then washed with PBS. Subsequently, the cells were replaced with fresh serum-free DMEM and treated with octafluoroserine, which was treated with insulin, a hypoglycemic hormone. Glucose uptake was measured by measuring the amount of glucose contained in the medium and calculating the amount of glucose transferred into the cells. The degree of glucose uptake into the muscle cells of the calculated octafluoroserine was shown in FIG.

As a result, as shown in Fig. 6, when the muscle cells were treated with octafluoroserine (concentration treatment: 6.25 to 50 μM) for one hour, the glucose utilization rate was increased in a concentration-dependent manner, It has been shown that the augmentation increases glucose uptake in muscle cells. In addition, 50 μM of octafluoroserine, the most abundant glucose uptake, was measured for glucose uptake at the treatment time. As a result, the glucose uptake was maximized at 2 hours after treatment and showed glucose absorption similar to that of insulin.

Therefore, the inventors of the present invention confirmed that octafluoroserine of the present invention has an effect of accelerating glucose uptake into muscle cells.

<5-3> Into muscle cells  For the mechanism of glucose uptake Western blot  analysis

Cells used in the experiment described in <5-2> were collected and the cells were eluted with a lysis buffer. Then, the cell eluate was separated into cell membranes and cytoplasm, and protein contents were measured. Then, GLUT4 expression The expression of phospho-Akt and phospho-AMPK in anti-glut4 antibody and anti-phospho-Akt (Ser 473) and anti-phospho-AMPK (Thr 172) Western blot method was performed.

As a result, as shown in Fig. 7, octafluoroserine was found to increase the amounts of Akt (insulin-dependent), AMPK (insulin-independent) and GLUT4 protein expression, which are essential for glucose uptake into muscles. Also, it was confirmed that protein expression decreased when pretreated with Akt and AMPK inhibitor, respectively. These results suggest that glucose uptake into the muscle cells of octafluoroserine is mediated by the activation of Akt and AMPK, and GLUT4, which is a glucose transporting agent, is transported to the cell membrane and glucose is absorbed into the cell. In conclusion, octafluoroserine activates both Akt and AMPK, thus GLUT4 is transferred to the cell membrane and glucose uptake into the muscle appears.

< Example  6>

I have a type 2 diabetic animal Octafloresol Ai  Blood glucose lowering effect measurement

Five - week - old male ICRC57BL / 6 db / db mice were purchased from the central laboratory animal and used for the experiment after 4 weeks of normal diet. The experimental group was randomly assigned to the control group, octafloresol A 2.5 mg / kg BW group, and octafloresol A 5 mg / kg BW group. Experiments were performed for a total of 2 weeks, and the Octafloresol A group was intraperitoneally administered at the above doses once a day. The temperature and humidity of the breeding room were maintained at 20 ± 2 ° C and 50 ± 10%, and the darkness and the humidity were adjusted at intervals of 12 hours. Weight and fasting blood glucose were measured at regular intervals during the experiment. Fasting blood glucose was measured by fasting blood / db mice twice a day for 12 hours during the night, and then blood was collected from the tail vein and blood glucose was measured with a simple blood glucose meter. After 2 weeks of experimentation, blood was collected from db / db mice and centrifuged to separate the plasma. Insulin concentrations were measured by ELISA using Rat / Mouse Insulin ELISA kit.

As a result, as shown in Fig. 8, it was found that the octafluoroside-treated group in the type 2 diabetic rats showed a continuous decrease in blood glucose compared to the control group. From these results, it was confirmed that octafluoroserine had an effect of lowering the blood glucose increase even in the type 2 diabetic model rats.

Furthermore, the present inventors measured the concentration of insulin in the blood after 2 weeks from the start of the experiment. As a result, as shown in Fig. 9, the insulin content in blood was lower in the octafloresol A group than in the control group, In the case of 5 mg / kg BW of floresal A, the content was about twice as low as that of the control group.

On the other hand, insulin resistance observed in most diabetic patients leads to impaired glucose tolerance and hyperinsulinemia, and hyperinsulinemia is known to be an important cause of lipid metabolism disorder, hypertension and coronary artery disease.

Therefore, the present inventors have found that the octafluoroserine compound of the present invention has an insulin resistance improving effect through the results of the present invention as described above.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (7)

A pharmaceutical composition for lowering blood glucose comprising an octafluoroserine compound represented by the following formula as an active ingredient;

. The method according to claim 1,
Wherein the compound is derived from Ishige foliacea. The method according to claim 1,
The above-mentioned octafluoroserine compound is a compound
Adding methanol to the wort powder to obtain a widened methanol extract;
Adding water and ethyl acetate to the obtained extract to obtain an ethyl acetate fraction;
Ethyl acetate fraction is carried out by performing silica gel chromatography using chloroform and a methanol solvent;
The fraction obtained through silica gel chromatography was subjected to Sephatex LH-20 column chromatography using a methanol solvent; And
Wherein the fraction obtained through Sephatex LH-20 column chromatography is further subjected to high performance liquid chromatography (HLPC). The method of claim 3,
Wherein the chloroform and the methanol solvent are mixed at a ratio of 50: 1 to 1: 1 in the course of performing silica gel chromatography using the chloroform and the methanol solvent. The method of claim 3,
Methanol used as an elution solvent used in the Sephatex LH-20 column chromatography was 100% methanol, and methanol used as a solvent for performing high performance liquid chromatography (HLPC) was 30-40% methanol A pharmaceutical composition for lowering blood glucose levels. The method according to claim 1,
Wherein said octafluoroserine compound has a digestive enzyme inhibitory action of carbohydrate, a glucose absorption promotion action in muscles and an insulin resistance improving action. A health functional food for preventing or ameliorating diabetes comprising the octafluorosulfan compound of claim 1 as an active ingredient.

Images