KR20150115973A - Composition for treating diabete and diabete-induced complication containing an extract from Aster sphathulifolius - Google Patents

Abstract

Provided is a composition for preventing and/or ameliorating diabetes or diabetic complications containing an extract of Aster sphathulifolius of the extract as an active ingredient. More specifically, the extract of Aster sphathulifolius exhibits effects of reducing blood sugar, and outstanding activities for ameliorating insulin resistance and preventing diabetic complications due to high blood sugar. Therefore, the present invention can be useful for a composition for preventing or treating major symptoms of diabetes and diabetic complications, functional food, pharmaceutical composition for regulating drop in blood sugar level, or health food for regulating drop in blood sugar level.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing or ameliorating diabetes and diabetic complications,

  The present invention relates to a composition for preventing and / or ameliorating diabetic and diabetic complications containing an extract of Aster sphathulifolius as an active ingredient.

Diabetes mellitus is a chronic disease that has been attracting attention worldwide because of the recent increase in the level of living standards of modern people and the increase in the longevity of human life.

 Diabetes mellitus is a metabolic syndrome characterized by a deficiency of insulin hormone or insulin resistance in the beta cells of the pancreas, as well as hyperglycemia resulting from both of these defects. Such diabetes can be divided into insulin dependent diabetes mellitus (IDDM, Type 1) and noninsulin dependent diabetes mellitus (NIDDM, Type 2) caused by insulin resistance and insulin secretory damage. In both type 1 and type 2 diabetes, diverse complications such as heart disease, bowel disease, ophthalmic disease, neurological disease, and stroke arise because of increased blood glucose and insulin levels for a long time, resulting in chronic neurological disease and cardiovascular disease And short-term hypoglycemia and hyperglycemia reaction cause acute complications.

 Diabetes mellitus, chronic hyperglycemia, as well as lipid metabolism, lipid and protein metabolic disorders are also caused. The condition is diverse and is directly caused by hyperglycemia, and includes diabetic peripheral neuropathy, diabetic retinopathy, diabetic nephropathy, diabetic cataract, keratosis, and diabetic atherosclerosis in the retina, the kidney, the nervous system, and the cardiovascular system. The number of diabetic patients is steadily increasing due to changes in lifestyle such as overeating, lack of exercise, westernized eating habits, stress, drinking and smoking.

 According to the Korean Diabetes Association, the prevalence of diabetes in Korean adults is estimated to be at least 10%, ranging from at least 3 million to at most 5 million, and the trend is steadily increasing. As the life expectancy of diabetic patients is prolonged, diarrhea due to diabetic complications and their deaths are becoming important social problems. Diabetic complication refers to a serious disease state caused by various functional or morphological changes in various tissues of the body depending on the degree of diabetes exposure. It usually occurs in eyes, kidneys, heart, nervous tissue, blindness, chronic heart failure, myocardial infarction, It is the most important to prevent the outbreak because it causes fatal diseases such as cutting.

 Diabetes mellitus is medication, exercise and diet, and insulin medicines and various blood glucose drugs are used depending on the patient's symptoms. However, diabetes is a complex disease characterized by overgrowth of glucose in the liver, insulin resistance, and decreased glucose tolerance in muscle and adipocytes. Therefore, specific treatment alone can not prevent the occurrence of various side effects. Because of the use of insulins and chemicals, drug therapy is a constant problem for drug side effects and patient resistance. Recently, diabetes treatment has been used to treat diabetes, And to further prevent and treat diabetic complications. Therefore, it is necessary to develop a material that effectively prevents diabetes and its complications from natural materials that are advantageous in terms of toxicity, and to scientifically verify the efficacy.

Accordingly, the present inventors have found that the present invention has excellent effects in the treatment of diabetic and diabetic complications of the colonies, and completed the present invention.

 Accordingly, one example of the present invention provides a composition for preventing and / or ameliorating diabetic and diabetic complications, which comprises at least one selected from the group consisting of extracts from seaweed as an active ingredient.

Hereinafter, the present invention will be described in more detail.

 There is provided a composition for preventing and / or ameliorating diabetic and diabetic complications, comprising the extract of sea bream and / or the above extract as an active ingredient.

 Aster spathulifolius is a perennial plant of the dicotyledonous spiny lily of the valley, also known as the seashore country. The stem is somewhat lignified, the branches are split a lot and grows diagonally and grows up to 30 ~ 60 cm in height. Leaves are alternate, but they look like inverted egg. They are gathered from bottom and thick. They are whitish on both sides and look white. Leaf edges are plain or have little saw teeth and spatulate. Flower is bloomed from July to November, light purple, with head hairs on the end of the branch. The head is hemispherical. The hairs are hairy and arranged in 3 rows. The fruit is mature in November. The hair is light brown and has a thin hair.

There is no research result that has been used for two purposes, but it is also used as an ornamental bonsai utilizing the strong vitality and lignification which does not die in the winter, while the young leaves are used for food, the outpost is used for diabetes, cystitis and the like. L-6-deoxyidopyranoside and labda-7, 14-dien-13 (R) -ol - Terpene glycosides such as L-6-deoxyidopyranoside, pigment of flowers, etc., and studies on other components of sea water are rarely performed. Currently, the patent for the improvement of blood lipid and the anti-obesity effect on the extract of the ground surface region has been registered at the Kyung Hee University Research Team (Kyung Hee University Industry-Academy Collaboration Team). (Patent No. 10-0919323 2009.09.21. (Korea), Patent ZL200680008949. X 2012.06.20. (China) In this patent, the content of terpene compounds such as germodrome and alpha-spinosterol glycosides in Korean sea liquor extract as well as ethanol or water extract of seaweed is greatly increased to improve blood lipid and anti-obesity effect Therefore, the present invention aims to demonstrate the effect of diabetes and diabetic complications of the colonies which have not yet been carried out in the present invention.

 The present invention provides a therapeutic effect of dietary extract composition for treating diabetes and diabetic complications.

 Diabetic complications are diabetic complications that lead to hyperglycemia and protein reactions that irreversibly progress to produce advanced glycation endproducts (AGEs) that cross-link with other proteins in the blood or tissue. Specifically, in the present invention, the diabetic complication having a therapeutic effect on the extract of sea bream may be diabetic vascular disorder, diabetic neuropathy or diabetic infection, and preferably diabetic retinopathy, diabetic cataract ), Diabetic nephropathy, diabetic neuropathy, diabetic vascular complications, and the like, more preferably diabetic retinopathy, diabetic cataract or diabetic nephropathy.

 Alpha-glucosidase is the most important carbohydrase and promotes the degradation of the glucose linkage at the end of the carbohydrate digestion process. Alpha-glucosidase inhibitors have the effect of lowering digestion and absorption rate of dehydrated products such as twelve fats, and can be used for the prevention and treatment of adult diseases such as diabetes, obesity, and fibrinolysis.

Aldose reductase is one of the mechanisms of diabetic complications, and the increase of polyol pathway is the mechanism of cell damage. Aldose reductase is an enzyme involved in the conversion of glucose from sorbitol to fructose in the polyol pathway. In hyperglycemic conditions, changes in glucose metabolism occur in the cell. When excess glucose enters the cell, it is converted to sorbitol by aldose reductase and converted to fructose by sorbitol dehydrogenase. The Km value (70 mM) of aldose reductase to glucose is high in the normal glucose state, and the concentration of sorbitol in the cell is very low. However, when the intracellular glucose concentration is increased by hyperglycemia, the sorbitol concentration in the cell is increased, and the sorbitol dehydrogenase reaction rate is slow, so that sorbitol accumulates in the cells. Ultimately, the high osmotic pressure of sorbitol accumulated in the cells expands the cells and damages the cells, resulting in complications such as cataract, diabetic neuropathy and retinopathy. Therefore, this study can be used for the prevention and treatment of diabetic complications.

The diabetic and diabetic complication prevention and therapeutic activities of the present invention are based on inhibition of the formation of advanced glycation endproducts (AGEs), inhibition of aldose reductase and inhibition of alpha-glucosidase . In addition, the in vitro test confirmed the possibility of prevention of diabetes and diabetic complications in the colonies, and then it was shown that the C57BL / KsJ-leprdb / leprdb (db / db) mice of the type II diabetes model had excellent effects on diabetic and diabetic complications .

The sea plant extract according to the present invention has an excellent effect of inhibiting blood glucose increase (see Table 1 and Fig. 1). Alpha-glucosidase is a digestive enzyme present in the villi of the small intestine and serves to hydrolyze disaccharides and small sugars into monosaccharides which are digestion and absorption state of carbohydrates. Therefore, alpha-glucosidase inhibitors can inhibit the increase of blood glucose level by inhibiting the hydrolysis of carbohydrates (lower digestion and absorption rate) at the upper part of the plant including the duodenum. Therefore, for the prevention and treatment of adult diseases such as diabetes, obesity and hyperglycemia Can be used.

As a result of testing the inhibitory activity against the final glycation endproduct of Korean seaweed extract according to the present invention, it was found to have an inhibitory effect on the final glycation end product formation (see Table 2 and FIG. 2). The final glycation end product is formed by non - enzymatic glycation reaction through binding of sugar to protein and schiff base and amylose product. However, in a chronic hyperglycemic state, perhaps a lipid product is reversibly accelerated by a irreversible reaction that produces a final glycation product and is mass-produced and accumulated in tissues and serum. Thus, the production of the final glycated product is one of the major causes of severe diabetic complications. It is known that the concentration of the final glycated product in the blood of patients with type 2 diabetes is highly correlated with the presence or severity of cardiovascular disease and also correlates with various animal model studies. The end glycation products produced by non-enzymatic glycation of chronic hyperglycemia and biologic proteins are one of the major causes of diabetic complications. Diabetic complications often occur despite the normal recovery of blood sugar. The final glycation product is a protein glycosylation product accumulated in tissues in proportion to the concentration of hyperglycemia, and exhibits a specific absorbance and fluorescence. Especially, amino groups of proteins cause cross-link reaction with each other. Once formed, the final glycation end product is not degraded even when blood glucose is normally regulated, and accumulates in tissues to abnormally change the structure and function of the tissue, and is known to be involved in atheroma atherosclerosis, DNA mutation, senescence, Alzheimer's disease, and diabetic complications have. Since the toxicity of aminoguanidine, which is known as an inhibitor of the final glycation end product, has been increased, it is necessary to develop a safe natural product which does not have toxicity.

Diabetes causes hyperglycemia in the blood, leading to increased oxidative stress and ROS. The produced ROS promotes the production of toxic aldehydes, and the produced toxic aldehydes activates the aldose reductase, producing sorbitol and fructose through the polyol pathway, about 2 to 4 times as much as glucose in the normal state. As a result of testing the inhibitory activity against algal reductase of Korean seaweed extract according to the present invention, it was shown to have an aldose reductase inhibitory effect (see Table 3 and FIG. 3).

The extract of sea bream according to the present invention has excellent antioxidant activity (see Table 4 and Fig. 4). DPPH (1,1-diphenyl-2-picrylhydrazyl) scavenging effect was tested to test the antioxidant effect of the composition of the present invention. DPPH is a chemically stable compound with free radicals and is widely used to confirm antioxidant ability when it is contacted with a substance having antioxidant activity. The electron donating action using DPPH is used for the purpose of inhibiting lipid oxidation in foods by donating electrons to free radicals and is being used in the body to suppress aging by free radicals.

The extract of the sea thalamus according to the present invention can be used for fasting blood glucose measurement and intraperitoneal glucose tolerance test every week through C57BL / KsJ-leprdb / leprdb (db / db) diabetic mouse and non-diabetic db / As a result of measuring the insulin resistance test, the fasting blood glucose level was improved (see FIG. 5) as compared with the diabetic group at the 3rd week, and blood glucose was measured for 0 to 120 minutes by the intraperitoneal glucose tolerance test before the autopsy. It was confirmed that the administration of the extract returned to the initial blood glucose level at 120 minutes (see FIG. 6). In the insulin resistance test, the insulin resistance was also found to be effective at 60 minutes since the blood glucose level did not rise as in the normal group. HbA1c and serum insulin concentrations were also significantly decreased by the addition of sea bass extracts, indicating that the sea bream extract was effective in alleviating and improving diabetic symptoms (see Table 6).

 The composition of the present invention can be used as an active ingredient in addition to the sea bass extract as well as nutrients, vitamins, electrolytes, flavors, colorants, enhancers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickening agents, pH adjusting agents, , A glycerin, an alcohol, a carbonating agent used in a carbonated drink, and the like. The components can be added independently or in combination. The content of the additional ingredient may be added in the range of 0.1 to 20 parts by weight per 100 parts by weight of the sea bream extract or fraction or 100 to 10,000,000 parts by weight per 100 parts by weight of lycoricidine, but is not limited thereto.

 The content of the Korean seaweed extract in the composition may be appropriately adjusted according to the symptom of the disease, the progress of the symptoms, the condition of the patient and the like, for example, 0.0001 to 99.9% by weight, preferably 0.0001 to 50% But is not limited thereto. The content ratio is a value based on the dried amount from which the solvent is removed.

 The composition may further comprise a pharmaceutically acceptable carrier, excipient and diluent used routinely in the preparation of a pharmaceutical composition. The composition may be formulated into a powder, granule, tablet, capsule, suspension, emulsion, syrup, aerosol Oral formulations, external preparations, suppositories, or sterile injection solutions, and the like.

 When the composition is formulated, it is prepared using a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, or an excipient usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations may include at least one excipient and / or lubricant. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, and the like.

 The preferred dosage of the composition varies depending on the condition and the weight of the patient, the degree of the disease, the drug form, the administration route and the period, but can be appropriately selected by those skilled in the art. For a more preferable effect, the dose of the composition of the present invention is preferably 0.1 mg / kg to 20 mg / kg per day based on the active ingredient, but is not limited thereto. The administration can be carried out once a day or in divided doses. The compositions of the present invention may be administered to a mammal, including a human, in various ways. All modes of administration may be expected, for example, by oral, intravenous, intramuscular, subcutaneous injection, and the like. The pharmaceutical dosage form of the composition of the present invention may be used in the form of a pharmaceutically acceptable salt of the active ingredient, and may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable combination.

 In another example, there is provided a composition for preventing and / or ameliorating diabetic and diabetic complications including decomposed plant extract as described above. In the present invention, the term " food " means a natural product or a processed product containing one or more nutrients, preferably a state of being able to be eaten directly through a certain degree of processing, , Health functional foods, beverages, food additives and beverage additives. Examples of the food include various foods, beverages, gums, tea, vitamin complex, and functional foods. In addition, in the present invention, the food may contain special nutritional foods (e.g., crude oil, spirits, infant food, etc.), meat products, fish products, tofu, jelly, noodles (Such as soy sauce, soybean paste, hot pepper paste, mixed sauce), sauces, confectionery (eg snacks), dairy products (eg fermented milk, cheese), other processed foods, kimchi, pickled foods But are not limited to, fruits, vegetables, beverages, beverages, fermented beverages, ice creams, etc.), natural seasonings (eg, ramen soup, etc.), vitamin complexes, alcoholic beverages, . The health functional food, beverage, food additive or beverage additive can be produced by a usual production method.

 The food composition may comprise a pharmaceutically acceptable food-aid additive and may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of a functional food.

 In addition, in the food, the amount of the exported sea extract may be 0.00001 wt% to 50 wt% of the total food weight, and when the food is a beverage, 0.001 g to 50 g, Preferably 0.01 to 10 g, but is not limited thereto.

The composition containing the extract of sea flower according to the present invention is a composition derived from a natural product which is excellent in the therapeutic effect of diabetic and diabetic complications but has no toxicity and side effects, and thus can be safely applied to the treatment of diabetic and diabetic complications.

Fig. 1 relates to an alpha-glucosidase inhibitory effect test of P. aeruginosa extracts.
Figure 2 relates to a test for the inhibitory effect of the final glycation products (AGEs) of offshore extracts.
Fig. 3 relates to a test for the inhibition of Aldose reductase of Korean marine extracts.
Fig. 4 relates to a test for antioxidative effects of DPPH extracts from P. aeruginosa extracts.
FIG. 5 is a chart showing the body weight measurement once a week during the breeding of the C57BL / KsJ-db / db Mouse by administering the extract of the sea bream.
FIG. 6 shows the glucose tolerance test (intraperitoneal glucose tolerance test) effect on the C57BL / KsJ-db / db Mouse according to the administration of the extract from the sea bream.
FIG. 7 shows the effect of insulin resistance test according to administration of C57BL / KsJ-db / db mouse cotyledon extract.
8 shows fasting blood glucose levels once a week according to the administration of C57BL / KsJ-db / db mouse cotyledon extract.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are merely illustrative of the present invention, and the scope of the present invention is not limited by these examples.

Material preparation

Aster sphathulifolius was collected from Ulleungdo Island and used as a sample. DPPH (1,1-diphenyl-2-picrylhydrazyl), dimethyl sulfoxide (DMSO), albumin from bovine serum, methylglyoxal and aminoguanidine were purchased from Sigma (USA) -glucosidase was purchased from Wako (Japan), and all the reagents used for the analysis were Express.

Sample preparation example

The extract of the present invention was prepared by adding various solvents (water, 10%, 30%, 50%, 95% ethanol) 10 times as much as the dry weight of 1000 g and extracting it for 3 hours at 50-90 on an aqueous solution, To obtain an extract. The extraction process is repeated three times. The obtained extract is concentrated at a reduced pressure of 50 or less and freeze-dried to obtain a powdery extract.

Example 1. Alpha-glucosidase inhibition test

 -glucosidase activity, p-nitrophenyl-Alpha-D-glucoside is converted to alpha-D-glucose and p-nitrophenol by alpha-glucosidase, and the final product is yellow. Yellow can be obtained by measuring the final absorbance using a spectrophotometer at a wavelength of 405 nm.

 The samples used in this study were dissolved at a concentration of 1 mg / mL. -Glucosidase was dissolved in phosphate buffer (pH 7.0) to make an enzyme solution. 10 L of sample was added to 60 L of enzyme solution, pre-incubated at 37 for 15 min, and p-nitrophenyl-D-glucopyranoside was dissolved in phosphate buffer ), 30 L of a substrate solution was added and the absorbance was measured at 405 nm. Acarbose was used as a positive control, and each sample was repeated three times and averaged.

The obtained results are shown in Table 1 and Fig. The ethanol extract of 95% ethanol extract showed the highest inhibition at 16.79% at 1 mg / mL, followed by the 50% ethanol extract at 16.79% inhibition. 50% and 95% were significantly different from each other. Therefore, it is considered that the country can effectively prevent blood sugar from rising sharply.

Sample Concentration
(mg / mL) Inhibition
(%) IC ₅₀
(mg / mL) Acarbose 1.6
2.5
3.3 46.360.92
70.622.23
79.710.48 0.2620.02 Water ex. One 7.582.12 - 10% EtOH ex. One 8.891.78 - 30% EtOH ex. One 11.071.08 - 50% EtOH ex. One 16.790.71 - 95% EtOH ex. One 19.161.97 -

Example 2. Test for the inhibitory effect of the final glycation products (AGEs)

 In this Example 2, in order to test the therapeutic effect of diabetic complication, the effect of inhibiting the final glycation end products (AGEs) of the extract from the sea bream extract was measured by Blank and the increase of fluorescence was measured by the 24 hour short term detection method using methylglyoxal Respectively.

 A sample (10 mg / mL of sea tangle extract obtained from the above-mentioned sample preparation) dissolved in DMSO (dimethyl sulfoxide) was added to BSA (5 mg / mL) and methyl glyoxal (2 mg / mL) and 50 mM phosphate buffer To adjust the total volume to 5 mL. After incubation at 37 for 24 hours, the absorbance (excitation: 355 nm, emission: 460 nm) of the sample before and after the reaction was measured using a spectrofluorometer (Fluoroskan Ascent FL, Theremo Scientific, USA). The inhibition rate of the absorbance was calculated by comparing the absorbance increase value of the sample based on the absorbance increase value of the control group.

The formula = {1- (S1-S0) / (C1-C0)} * 100

(S0: sample 0hr absorbance response value, S1: sample 24hr absorbance response value, C0: control 0hr absorbance response value, and C1: control 24hr absorbance response value) were used. In addition, fluorescence inhibition rates were compared using aminoguanidine (Sigma Chemical Co., St. Louis, Mo., USA) as a standard substance known to have an inhibitory effect on the final glycation end product.

 The obtained results are shown in Table 2 and Fig. As a result of measuring the degree of increase of fluorescence based on the blank and the control group, 50% ethanol extract showed the highest inhibition rate of 93.88% at the concentration of 200 g / In addition, IC50 values of the aminoguanidine and 50% ethanol extracts compared with the positive control group were about 65.09 and 89.38 g / mL, respectively, indicating that the extract from Korea has similar effects to that of the positive control group. Respectively.

Sample Concentration
(g / mL) Inhibition
(%) IC ₅₀
(g / mL) Aminoguanidine 50
100
200 42.302.56
65.711.01
95.440.31 0.2620.02 Water ex. 200 49.001.59 - 10% EtOH ex. 200 49.401.13 - 30% EtOH ex. 200 54.251.96 - 50% EtOH ex. 50
100
200 30.760.51
58.971.00
93.881.59 89.380.71 95% EtOH ex. 200 65.361.56 -

Example 3. Aldose reductase inhibition test

 In Example 3, in order to test the therapeutic effect of diabetic complication, the inhibitory effect of Aldose reductase, which measures the decrease rate of NADPH absorbance, was confirmed using a rat lens lens as an enzyme and NADPH and DL-glyceraldehyde as a substrate.

 - Preparation of enzyme source: A rat lens with a weight of 200 g or more was extracted and homogenized in ice with 250 L of 0.05 M sodium buffer (pH 6.8) per lens according to the wet weight of the lens. This was centrifuged at 7500 rpm for 30 minutes (centrifuge UNION 55 R Hanil, Korea), and the supernatant was used as an enzyme source.

 - Aldose reductase inhibitory activity measurement: Aldose reductase inhibitory activity was tested by modifying the method used by Haymanh and Kinoshita. To the 530 L 0.05 M potassium phosphate buffer (pH 7.0), 160 L of the enzyme source, 100 L of 1.6 mM NADPH and 10 L of the fraction (1 mg / mL) were added and finally 100 L of 0.1 M DL-glyceraldehyde was added. Cells were reacted for 4.4 minutes to measure the decrease of NADPH absorbance at 340 nm. In addition, quercetin was used as a standard substance known to inhibit Aldose reductase, and the rate of decrease in absorbance was measured and compared with the fractions. The concentration of the sample (IC50), which shows a decrease in the absorbance at 50% of the control, was expressed, and each sample was repeated three times and averaged.

 The obtained results are shown in Table 3 and FIG. As a result of measuring the degree of increase of absorbance based on the blank and the control group, the highest concentration of the extract was found to be 86.44% at the concentration of 11.11 g / mL of 50% ethanol extract. The IC50 values of quercetin and 50% ethanol were about 2.28 and 4.84 g / mL, respectively. These results suggest that the Korean extract has a therapeutic effect to prevent diabetic complications.

Sample Concentration
(g / mL) Inhibition
(%) IC ₅₀
(g / mL) Quercetin 0.56
5.56
11.11 23.731.56
62.711.60
84.752.48 2.280.21 Water ex. 11.11 72.881.46 - 10% EtOH ex. 11.11 57.631.16 - 30% EtOH ex. 11.11 49.151.45 - 50% EtOH ex. 2.78
5.56
11.11 15.250.89
69.491.15
86.442.20 4.840.08 95% EtOH ex. 11.11 47.462.09 -

Example 4. Antioxidant effect test using DPPH

 DPPH is a chemically stable compound with free radicals and is widely used to confirm antioxidant ability when it is contacted with a substance having antioxidant activity. Thus, in Example 4, the free radical scavenging effect (hydrogen donating ability to DPPH) of seaweed extract was evaluated using DPPH, and the antioxidative effect was evaluated.

 The extracts from sea bream were prepared by diluting with dimethyl sulfoxide (DMSO). To a 96-well microplate, 270 L of 5 10 -4 M DPPH solution (dissolved in 99% ethanol) was added to 30 L of the thus-prepared constant concentration sample, and the mixture was mixed well and left at room temperature for 30 minutes. The resulting reaction solution was measured by electron donating ability at an absorbance of 570 nm. The inhibitory activity of each fraction was calculated from the reduced absorbance versus the positive control.

 Calculation formula = {1- (control / sample)} * 100

 At this time, ascorbic acid (Showa Chemicals Inc., Japan), which is known to have an antioxidative effect, was used as a positive control for the activity comparison, and DPPH radical scavenging activity was measured by the same method as described above.

The results of comparison of DPPH radical scavenging ability of Korean seaweed extracts are shown in Table 4 and FIG. 3 times repeatedly.

 As a result of measuring the degree of increase of absorbance based on the blank and the control group, 50% ethanol extract showed a highest radical scavenging ability at a concentration of 100 / mL of 82.38%.

Sample Concentration
(g / mL) Inhibition
(%) IC ₅₀
(g / mL) Ascorbic acid 1.6
2.5
3.3 48.803.27
60.333.46
77.551.37 1.730.21 Water ex. 100 50.441.13 - 10% EtOH ex. 100 56.830.95 - 30% EtOH ex. 100 51.073.63 - 50% EtOH ex. 25
50
100 28.131.23
45.402.06
82.380.12 55.871.72 95% EtOH ex. 100 38.550.61 -

In order to confirm the effects of prevention and treatment of diabetes and diabetic complications of the colonies proven through Examples 1 to 4 above, C57BL / KsJ-leprdb / leprdb (db / db) diabetic mice, representative animal models of type 2 diabetes, Respectively.

Example 5. Diabetic experimental animal and experimental design

 Male C57BL / KsJ-leprdb / leprdb (db / db) diabetic mice and non-diabetic db / m + mice with specific pathogen-free 5-week-old representative animal models of type 2 diabetes, (Jung-Ang Lab, Animal, Inc, Seoul, Korea). The mice were weighed for about 14 days or more and the weight and blood glucose were measured after the adaptation period, and mice (12 mice per group) suitable for the laboratory and not considered to be pathological findings were selected. Experimental animals were exposed to air at a temperature of 20.9 ~ 22.6, a relative humidity of 45 ~ 55%, a ventilation frequency of 10 ~ 12 times / hour, a total ventilation time of 12 hours (8: 00 ~ 20: 00) The mice were housed in a polycarbonate breeding box (278420200 mm, Three Shine, Jinshan Co., Ltd.) in the setting environment, and the normal control db / m + mouse used in this study had an initial body weight of 25.500.35 g and a db / db mouse 37.201.56 g.

The animals were divided into normal (db / m +), diabetic (db / db), and 200 mg / Kg sea bream extract groups (db / db-AS200) for 7 weeks. Each of the administered materials was dissolved in physiological saline and orally administered once a day at the same time, and the negative control was orally administered with purified water. The diet of the experimental animals was purchased by supplying the commercially available experimental animal, AIN-76, through the central experimental animal, and water was freely taken.

Example 6. Weight, Dietary Intake, and Moisture Intake Measurements

The animals were weighed once a week (FIG. 5), and diet and water intakes (Table 5) were measured once a week.

Sample Diet intake
(g / day / mouse) Water intake
(g / day / mouse) db / m + 2.770.30 ^b 3.090.35 ^c db / db 4.410.55 ^a 11.820.47 ^a db / db-AS200 3.940.49 ^a 8.961.00 ^b

The C57BL / KsJ-leprdb / leprdb (db / db) mice are known to exhibit clinical symptoms similar to those of type 2 diabetes, including obesity, obesity, insulin resistance, hyperglycemia and hyperinsulinemia due to mutations in the leptin receptor gene. Body weight gain and dietary intake were decreased in the group treated with sea bream extract compared to the db / db group, but there was no significant difference between the groups, and water intake was significantly different in all groups. The group is believed to improve the following clinical symptoms of diabetes:

Example 7. Glucose tolerance test (intraperitoneal glucose tolerance test, IPGTT), insulin tolerance test (ITT)

 - Glucose tolerance test (intraperitoneal glucose tolerance test): The mice were fasted for 12 hours one week before the end of the test, and blood glucose was measured. Immediately after intraperitoneal administration of 5% glucose (2 g / kg body weight) , 60, 90, and 120 minutes, blood glucose was measured by taking blood from the tail vein.

- Insulin tolerance test: The mouse was fasted for 12 hours one day before the end of the test, and blood glucose was measured. Subcutaneous administration of 5 unit / kg body weight insulin was performed. After 30 minutes, 5% glucose (2 g / kg body weight) Blood was collected from the tail vein at 15, 30, 45, 60, 90 and 120 minutes after the administration.

The mice were fasted for 12 hours at the end of the test in order to confirm the glucose tolerance test. Blood glucose was measured, and 5% glucose was intraperitoneally administered to blood samples taken at 0, 15, 30, 60, and 120 minutes, and blood glucose levels were measured. In the normal group, the blood glucose level at 30 minutes was the highest, and after 120 minutes, the glucose level returned to the normal blood glucose level. In contrast, diabetic group showed the highest blood glucose level after 30 minutes but did not return to the initial blood glucose level after 120 minutes. After 120 minutes, the blood glucose level in the group treated with extracts from Korea was found to be close to the initial blood glucose level. It is expected that administration of sea bream extract improves glucose tolerance.

The results of measuring the insulin resistance of the mice administered with the extract from the sea bream are shown in Fig. As a result of measuring blood glucose after administration of insulin and glucose to all the groups, compared with the diabetic group, insulin resistance was improved in the group treated with sea bream extract, the blood glucose level did not rise as in the diabetic group from 60 minutes, , And showed a significant difference at 120 min. Therefore, it was shown that administration of sea bream extract improves insulin resistance.

Example 8. Measurement of blood glucose, serum insulin, and glycated hemoglobin (HbA1c) concentration

 The blood glucose was measured once a week for 12 hours in the experimental animals, and then the blood was collected from the tail vein and the fasting glucose was measured using a blood glucose meter (Roche). Serum insulin levels were determined using an insulin ELISA kit (Milipore) for insulin determination in sera isolated from blood samples obtained after sacrifice of experimental animals. HbA1c was obtained by fasting 12 hours before the sacrifice of the experimental animals, and orbital blood was collected and the whole blood was measured with a glycated hemoglobin analyzer (HLC-723Hb G7, Tosoh).

 Fig. 8 shows the results of weekly blood glucose changes by administering the extract of Coleoptera japonica to C57BL / KsJ-leprdb / leprdb (db / db) mice. C57BL / KsJ-db / db mice showed no difference in blood glucose level between C57BL / KsJ-db / db + mice and their heterozygous C57BL / KsJ-db / db mice at 5 weeks of age. It is known that blood glucose increases with increasing resistance. In the db / m + group of normal group, normal fasting blood glucose level was confirmed for 7 weeks. In the db / db group of diabetic group, blood glucose tended to increase rapidly from 2 weeks after the start of the experiment. In addition, it was confirmed that the group administered with sea bream extract did not significantly increase the blood glucose level as compared with the diabetic group.

 As a result of using the ELISA kit to determine the serum insulin concentration, the insulin secretion was normal in the db / m + group and db / m + in the normal group, db group showed hyperinsulinemia. In addition, hyperinsulinemia was improved in the db / db group treated with sea bream extract. Therefore, it is considered that the extract from sea bream improves insulin resistance. The HbA1c value, which is widely used in diabetes research, is estimated by the value of glycated hemoglobin, which is widely used as the most basic index for expressing blood glucose control. As a result, (Table 6). The results are shown in Table 6. This indicates that there is an effect of decreasing blood sugar by administration of Korean seaweed extract.

 Insulin is the most important factor in the effective use of glucose in blood, muscle, liver, and fat tissue. Even if the same blood sugar level is high, blood insulin is high, meaning that many insulin is needed to maintain the same condition. It means that insulin action is falling, and this condition is called insulin resistance. Therefore, insulin secretion increases in type 2 diabetic patients, hyperinsulinemia can be induced, and the longer the duration of the disease, the worse the insulin secretion deteriorates and the blood glucose control deteriorates. In this study, it was suggested that the extracts from Korea were helpful for reducing insulin secretion and HbA1c concentration by oral administration of Korean seaweeds through type 2 diabetic rats.

Sample Serum insulin
(mg / dL) HbA1c
(%) db / m + 0.660.02 ^b 3.820.16 ^c db / db 2.420.57 ^a 8.200.99 ^a db / db-AS200 1.560.19 ^ab 6.830.64 ^b

Example 9. Serum biochemical test

Serum isolated from the blood samples collected on the day of the autopsy was analyzed by using a blood biochemical analyzer (KoneLab 20, Thermo Fisher SCIENTIFIC, Waltham, Finland) with the use of alanine aminotransferase (ALT), aspartate aminotransferase (AST), cholesterol (CHOL), LDL-choleterol LDL-C), HDL-cholesterol (HDL-C) and triglycerides (TG) were measured.

Sample ALT
(U / L) AST
(U / L) CHOL
(mg / dL) LDL-CHOL
(mg / dL) HDL-CHOL
(mg / dL) TG
(mg / dL) db / m + 33.514.85 ^c 95.398.77 ^b 136.074.66 ^b 11.381.83 ^b 113.354.50 ^a 53.343.32 ^a db / db 555.2550.60 ^a 414.1229.94 ^a 247.9138.56 ^a 31.985.49 ^a 132.3717.61 ^a 68.857.81 ^a db / db-AS200 384.0742.14 ^b 326.2937.42 ^a 209.5222.76 ^ab 22.012.15 ^ab 147.269.13 ^a 68.7311.74 ^a

In Example 9, sera of each experimental group were analyzed using a blood biochemical measuring device, and all of ALT, CHOL and LDL-CHOL were significantly decreased. ALT, which is used for liver function tests, is an enzyme mainly present in liver, and its activity is increased by hepatitis, hepatic necrosis and liver cirrhosis. In the present study, ALT levels were significantly decreased in the group treated with Korean seaweed extract compared to the diabetic group. In addition, AST is an enzyme present in the liver, heart, muscle, and kidney. When these tissues are damaged, the enzyme is liberated in the blood and the enzyme activity is high, and relatively high correlation with the degree of cell damage is widely used as an index of hepatitis and liver cirrhosis . AST also showed a tendency to decrease compared to the diabetic group.

 HDL-cholesterol lowers blood cholesterol levels and is effective in the prevention and improvement of arteriosclerosis

. In the present study, the tendency was increased by administration of sea bream extract, and LDL-cholesterol was significantly lower than that of diabetic group, suggesting that the risk of arteriosclerosis may be reduced.

Therefore, extracts from sea bream effectively lowered blood glucose levels in db / db mice, and this effect could be expected to improve diabetic complications as well as improve serum lipid levels.

Statistical processing

 Statistical analysis of all data was performed using the SAS statistical program (SAS institute, 1987). The results were expressed as means and standard errors per experiment group. The significance of the treatment groups was tested by Duncan's multiple range test at p0.05 level.

Claims (7)

An extract obtained by extracting the yeast from one or more members selected from the group consisting of water and straight chain or branched alcohols or mixtures of C1 to C4; and an extract obtained by dissolving the extract in water, one kind selected from the group consisting of hexane, methylene chloride, ethyl acetate and butanol And a fraction obtained by fractionation of the above-mentioned fraction as an active ingredient, and a pharmaceutical composition for the prevention and treatment of diabetic and diabetic complications The pharmaceutical composition according to claim 1, wherein the exporting country is extracted with water, an organic solvent or a mixed solvent thereof The pharmaceutical composition according to claim 1, wherein the diabetic complication is arteriosclerosis, cerebral infarction, cerebral thrombosis, myocardial infarction, hypertension, hyperlipidemia or obesity A health functional food for prevention and improvement of diabetic or diabetic complications containing extract from sea bream or an active ingredient thereof The health functional food according to claim 4, wherein the health functional food is a health functional food having any one of tablet, capsule, powder, granule, liquid, Improving the blood glucose level, improving the lipid metabolism and preventing and treating diabetic complications, or a health supplement containing the active ingredient The food according to claim 6, wherein the food is a food, a beverage, a gum, a tea, a vitamin complex,

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