KR101710276B1 - Composition for anti-diabete comprising ethanol extract of tuna heart - Google Patents

Abstract

The present invention relates to an anti-diabetic composition comprising an ethanol extract of a tuna heart as an active ingredient. According to the present invention, the ethanol extract of a tuna heart has activity of inhibiting PTP1B, -glucosidase and human recombinant aldose reductase, thereby being used for preventing or treating diabetes or diabetic complications. Also, the ethanol extract of a tuna heart is stable in the body, thereby being used as a functional health food material.

Description

[0001] The present invention relates to a composition for anti-diabetes comprising tuna heart ethanol extract as an active ingredient.

The present invention relates to a composition for anti-diabetes comprising an extract of tuna heart ethanol as an active ingredient.

Currently, Korea is experiencing rapid changes in the pattern of disease due to the rapid growth of Westernized dietary habits and nutrients, and chronic degenerative diseases such as arteriosclerosis, hypertension, cancer, obesity and diabetes are major causes of death .

In particular, the incidence of diabetes mellitus and its complications is increasing rapidly. The incidence of diabetes was less than 1% of the population in the 1970s. Since the 1990s, 17.2 people per 100,000 population have died from diabetes, and it is estimated that the incidence of diabetes will continue to increase in the future. Diabetes mellitus is a chronic metabolic disorder in which hyperglycemia and its metabolic disturbance persist for a long period of time. It is a chronic metabolic disorder in which insulin-dependent diabetes mellitus and insulin secretory ability, which are caused by an absolute deficiency of insulin due to dysfunction of the pancreas Langerhan's island β- Non-insulin dependent diabetes mellitus.

Diabetes mellitus is a typical chronic disease, and it is a chronic disease that causes microvascular complications such as retinas, kidneys and nerves, and macrovascular complications such as stroke, angina, myocardial infarction and peripheral vascular disease due to various metabolic disorders including glucose. Recently, the development of diabetes mellitus has led to an increase in the life expectancy of patients with diabetes mellitus and a rapid decrease in mortality due to acute metabolic complications. However, due to the increased incidence of diabetic chronic complications, diabetic complications are more severe than diabetes mellitus itself It is pointed out as a problem.

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 drug therapy uses insulin and chemicals, drug therapy is a constant problem for side effects and patient resistance due to the use of drugs. Therefore, recently, diabetes can be eaten and diabetes can be diarrhea, It is necessary to study for treatment.

Korea Patent No. 1126677 Korean Patent No. 1193085

Accordingly, the present inventors confirmed that the tuna heart extract has excellent effects in the treatment of diabetic and diabetic complications, and completed the present invention.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for preventing or treating diabetic or diabetic complications comprising an extract of tuna as an active ingredient.

Still another object of the present invention is to provide a method for preparing a tuna heart, comprising: (a) placing a tuna heart in an ethanol solvent; (b) extracting the tuna heart prepared in the step (a) and filtering the filtered tuna; And (c) concentrating the filtered tuna heart with a vacuum rotary concentrator to remove the ethanol solvent. The present invention also provides a method for producing tuna heart ethanol extract.

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating diabetic or diabetic complications comprising the tuna heart ethanol extract prepared by the above method as an active ingredient.

Furthermore, it is an object of the present invention to provide a functional health food for preventing or ameliorating diabetic or diabetic complications comprising the tuna heart ethanol extract prepared by the above invention as an active ingredient.

In order to accomplish the above object, the present invention provides a pharmaceutical composition for preventing or treating diabetic or diabetic complication comprising an extract of tuna as an active ingredient.

In one embodiment of the present invention, the tuna may be a heart, a fin or a bouillon.

In one embodiment of the present invention, the diabetic complication may be diabetic retinopathy, diabetic cataract, diabetic nephropathy, diabetic neuropathy, diabetic vascular complication.

(A) placing a tuna heart in an ethanol solvent; (b) extracting the tuna heart prepared in the step (a) and filtering the filtered tuna; And (c) concentrating the filtered tuna heart with a vacuum rotary concentrator to remove the ethanol solvent.

In one embodiment of the present invention, the ethanol solvent of step (a) may be mixed with water and ethanol at a volume ratio of 3: 7.

In one embodiment of the present invention, the extraction of step (b) may be repeated three times at a temperature of 60 to 80 ° C for 1 to 15 hours.

The present invention also provides a pharmaceutical composition for preventing or treating diabetic or diabetic complications comprising the tuna heart ethanol extract prepared by the above method as an active ingredient.

Further, the present invention provides a functional health food for preventing or ameliorating diabetic or diabetic complications comprising the tuna heart ethanol extract prepared by the above method as an active ingredient.

The tuna heart ethanol extract according to the present invention has an activity of inhibiting PTP1B,? -Glucosidase and human recombinant aldose reductase, and thus can be effectively used for the prevention or treatment of diabetic or diabetic complications. In addition, the tuna heart ethanol extract of the present invention is a natural substance and stable in the body and can be used as a material for functional health food.

Figure 1 shows the DPPH radical scavenging activity of 70% ethanol extract of tuna heart.
FIG. 2 shows the ABTS radical scavenging activity of 70% ethanol extract of tuna heart.
FIG. 3 shows the ONOO ^- radical scavenging activity of the 70% ethanol extract of tuna heart.
FIG. 4 shows the result of measuring the total phenol content of 70% ethanol extract of tuna heart.
FIG. 5 shows the results of confirming the degree of PTP1B inhibition of the 70% ethanol extract of tuna heart.
FIG. 6 shows the results of confirming the degree of suppression of? -Glucosidase in the 70% ethanol extract of tuna heart.
FIG. 7 shows the results of confirming the degree of suppression of human recombinant aldose reductase in the 70% ethanol extract of tuna heart.

The present invention relates to a novel use of a tuna heart extract, and is characterized by providing a composition for preventing or treating diabetes and diabetic complications comprising an extract of tuna heart ethanol as an active ingredient.

Tuna ( Katsuwonus pelamis ) are seaweed meat with sea mackerel and mackerel, and are divided into two types: northern bluefin tuna in the northern hemisphere and southern bluefin tuna in the southern part of the three oceans. In December ~ February, the highest level of fat content is obtained, and it is eaten with sushi, sushi, etc. In addition, it is not only nutritionally superior to high protein foods, but also has various physiological activities such as reduction of blood cholesterol concentration, prevention of arteriosclerosis, reduction of cerebrovascular disease, restoration of heart disease and anticancer activity.

In addition, fish muscles contain w-3 polyunsaturated fatty acids such as EPA and DHA, and are effective in preventing hypertension, improving blood flow, preventing cranial nerve and myocardial infarction, preventing cancer and preventing senile dementia . EPA, which is one of w-3 polyunsaturated fatty acids, is widely used for prevention of diseases of the circulatory system such as thrombosis and atherosclerosis which have a function of significantly lowering the concentration of cholesterol and neutral lipid in blood, inhibition of colon cancer cell growth and hepatocyte cell growth, Is known to have an effect of inhibiting growth of the cells.

In addition, the anticancer activity and immunological test results of ethanol extract of tuna are known to inhibit the proliferation of human intestinal cancer cells, leukemic and blastocysts.

As mentioned above, tuna with various physiological activities are required to develop new processed foods that can easily maintain the taste and nutrients of tuna as well as sashimi and sushi, which are major intake types. The tuna is mainly used in canning production. Due to the simplicity and socioeconomic changes of modern people seeking high functional food, demand for processed fish products using tuna is increasing. However, in the processing of fisheries such as tuna, about 30 ~ 35% of the total raw materials are produced as by-products, and only a part is used as a low-cost seasoning or intermediate material for food, and most of them are used as feed or waste. Tuna by-products include heart, fish, flesh, tail, blood, flesh, and gut.

The inventors of the present invention have conducted various studies to industrially utilize the tuna by-product, which is a tuna by-product, which has been abandoned in the past, and have been studying PTP1B,? -Glucosidase and human recombinant aldose reductase Of diabetic and diabetic complications.

Accordingly, the present invention can provide a composition capable of preventing or treating diabetic and diabetic complications including tuna heart extract as an active ingredient.

The tuna heart extract according to the present invention can be obtained by extracting and separating from natural substances by using extraction and separation methods known in the art. The 'extract' defined in the present invention can be obtained by extracting a tuna heart For example, crude extracts of tuna heart, polar solvent-soluble extracts, or non-polar solvent-soluble extracts.

As an appropriate solvent for extracting the extract from the tuna heart, any pharmaceutically acceptable organic solvent may be used, and water or an organic solvent may be used. For example, purified water, methanol alcohol having 1 to 4 carbon atoms, acetone, ether, benzene, chloroform (including methanol, ethanol, propanol, isopropanol, and butanol) various solvents such as chloroform, ethyl acetate, methylene chloride, hexane and cyclohexane may be used alone or in combination. More preferably, water or ethanol (alcohol) can be used.

As the extraction method, any one of the methods such as hot water extraction method, cold extraction method, reflux cooling extraction method, solvent extraction method, steam distillation method, ultrasonic extraction method, elution method and compression method can be selected and used. In addition, the desired extract may be further subjected to a conventional fractionation process or may be purified using a conventional purification method. There is no limitation on the method for producing the tuna heart extract of the present invention, and any known method can be used.

For example, the tuna heart extract contained in the composition of the present invention can be prepared into a powdery state by an additional process such as vacuum distillation, freeze-drying, or spray-drying, with the primary extract extracted by the hot water extraction or solvent extraction method described above have. Further, the primary extract can be further purified by using various chromatographies such as silica gel column chromatography, thin layer chromatography, high performance liquid chromatography and the like, You can get it.

Therefore, in the present invention, the tuna heart extract is a concept including all the extract, fraction and purified product obtained in each step of extraction, fractionation or purification, and a diluted solution, concentrate or dried product thereof.

A method for preparing a tuna heart extract according to an embodiment of the present invention will be described in more detail as follows.

The material used for the production of the tuna heart extract of the present invention was the tuna heart of the Dongwon F & B Changwon factory, which was processed during the processing of canned tuna, Remove the remaining non-edible portion. Since the heart of the tuna is attached near the dorsum, it can not be removed during the intestinal removal process. During the process of removing the head, the immature state of the heart is produced. The tuna heart was immersed in 5 times of purified water for 30 minutes, then washed with running tap water for 10 minutes, and then washed with water for 30 minutes. Thereafter, the tuna heart powder was pulverized using a Silent Cutter (Fatosa, S.A., Spain) and passed through a 20-mesh Test Sieve 20 mesh with 2.5 holes in the standard length and 2.54 cm in length. The pulverized heart was analyzed while cryopreserved below -20 ℃.

The tuna heart powder is mixed with a volume of water having a volume of about 3 to 5 times the dry weight and a mixed solvent having a volume ratio of ethanol to water ratio of 3: 7, at an extraction temperature of 70 DEG C for about 0.5 to 2 days, Preferably 1 to 5 times, preferably 3 times, by continuous extraction method such as hot water extraction, cold extraction, reflux cooling extraction or ultrasonic extraction for 1 day. The filtrate is filtered under reduced pressure, Deg.] C, preferably 40 to 70 [deg.] C, to obtain a 70% ethanol extract of tuna cores soluble in these mixed solvents.

The composition of the present invention comprising the tuna heart extract prepared by the above method as an active ingredient may be a pharmaceutical composition or a food composition.

The pharmaceutical composition of the present invention can be prepared by using pharmaceutically acceptable and physiologically acceptable adjuvants in addition to the above-mentioned active ingredients. Examples of the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, A lubricant or a flavoring agent can be used.

The pharmaceutical composition may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the above-described active ingredients for administration.

The pharmaceutical composition of the present invention can be prepared by using pharmaceutically acceptable and physiologically acceptable adjuvants in addition to the above-mentioned active ingredients. Examples of the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, A lubricant or a flavoring agent can be used.

The pharmaceutical composition may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the above-described active ingredients for administration.

The pharmaceutical composition may be in the form of granules, powders, tablets, coated tablets, capsules, suppositories, liquids, syrups, juices, suspensions, emulsions, drops or injectable solutions. For example, for formulation into tablets or capsules, the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Also, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included as a mixture. Suitable binders include, but are not limited to, natural sugars such as starch, gelatin, glucose or beta-lactose, natural and synthetic gums such as corn sweeteners, acacia, tracker candles or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum and the like. Acceptable pharmaceutical carriers for compositions that are formulated into a liquid solution include sterile water and sterile water suitable for the living body such as saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.

In one embodiment of the present invention, the tuna heart extract of the present invention may be contained in an amount of 0.1 to 10% by weight based on the total weight of the composition.

The composition of the present invention may also be a food composition. In addition to containing the tuna heart extract as an active ingredient, such a food composition may contain various flavors or natural carbohydrates as an additional ingredient such as a conventional food composition.

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. The above-described flavors can be advantageously used as natural flavorings (tau martin), stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.).

The food composition of the present invention can be formulated in the same manner as the above pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolates, foods, confectionery, pizza, ram noodles, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes, .

In addition to the tuna heart extract, which is an effective ingredient, the food composition can also contain flavoring agents such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and thickening agents (cheese, chocolate, etc.) Salts of alginic acid and its salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the food composition of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks.

The tuna heart extract as an active ingredient of the present invention is a natural substance and has almost no toxicity and side effects. Therefore, it can be safely used for long-term use for the prevention or treatment of diabetes and diabetic complications.

The present invention also provides a health functional food for controlling blood sugar which comprises a tuna heart extract as an active ingredient.

The health functional food of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, and rings for the purpose of controlling blood glucose.

In the present invention, the term "health functional food" refers to foods manufactured and processed using raw materials or ingredients having useful functions in accordance with Law No. 6727 on Health Functional Foods, Or to obtain a beneficial effect in health use such as physiological action.

The health functional foods of the present invention may contain conventional food additives and, unless otherwise specified, whether or not they are suitable as food additives are classified according to the General Rules for Food Additives approved by the Food and Drug Administration, Standards and standards.

Examples of the items listed in the above-mentioned 'food additives' include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as persimmon extract, licorice extract, crystalline cellulose, high color pigment and guar gum; L-glutamic acid sodium preparations, noodle-added alkalis, preservative preparations, tar coloring preparations and the like.

For example, the health functional food in tablet form may be prepared by granulating a mixture of tuna heart extract, an active ingredient of the present invention, with an excipient, a binder, a disintegrant, and other additives by a conventional method, Or the mixture can be directly compression molded. In addition, the health functional food of the tablet form may contain a mating agent or the like if necessary.

The hard capsule of the capsule form of the health functional food can be prepared by filling a normal hard capsule with a mixture of a tuna heart extract of the present invention and an additive such as an excipient and the soft capsule is prepared by adding a tuna heart extract to an excipient such as an excipient And filling the mixture with a capsule base such as gelatin. The soft capsule may contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative and the like, if necessary.

The ring-shaped health functional food can be prepared by molding a mixture of a tuna heart extract, an active ingredient of the present invention, an excipient, a binder, a disintegrant, and the like by a conventionally known method and, if necessary, Or it may be coated with a material such as starch, talc.

The granular health functional food can be prepared by granulating a mixture of tuna heart extract, excipient, binder, disintegrant and the like, which is an effective ingredient of the present invention, into granules by a known method, and if necessary, And the like.

The health functional food containing the tuna heart extract of the present invention as an active ingredient has an activity of inhibiting PTP1B,? -Glucosidase and human recombinant aldose reductase, as shown in the following examples, and is effective for controlling blood glucose.

The health functional food may be a beverage, a meat, a chocolate, a food, a confectionery, a pizza, a ramen, a noodle, a gum, a candy, an ice cream, an alcoholic beverage, a vitamin complex and a health supplement food.

The present invention also provides a method of preventing or treating diabetic and diabetic complications comprising administering to a mammal a tuna heart extract.

As used herein, the term " mammal " refers to a mammal that is the subject of treatment, observation, or experimentation, preferably a human.

As used herein, the term " therapeutically effective amount " refers to the amount of active ingredient or pharmaceutical composition that elicits a biological or medical response in a tissue system, animal or human, as contemplated by a researcher, veterinarian, The amount that induces the relief of the symptoms of the disease or disorder being treated. It will be apparent to those skilled in the art that the therapeutically effective dose and the number of administrations of the active ingredient of the present invention will vary depending on the desired effect. Thus, the optimal dosage to be administered can be readily determined by those skilled in the art and will vary with the nature of the disease, the severity of the disease, the amount of active and other ingredients contained in the composition, the type of formulation, and the age, The age, body weight, sex, diet, time of administration, route of administration and fraction of the composition, duration of treatment, concurrent medication, and the like. In the treatment method of the present invention, in the case of an adult, it is preferable to administer the tuna heart extract of the present invention at a dose of 1 mg / kg to 250 mg / kg once to several times a day.

In the treatment method of the present invention, the composition comprising the tuna heart extract of the present invention as an active ingredient can be administered orally, rectally, intravenously, intraarterially, intraperitoneally, intramuscularly, intramuscularly, transdermally, topically, And can be administered in a conventional manner.

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>

Tuna heart 70% Ethanol Crude extract  Manufacturing method

In the 70% ethanol extract of tuna heart used in the present invention, 100 g of the frozen powdered tuna heart was added to 300 ml of water and ethanol composition ratio of 3: 7, and the mixture was repeated three times at a constant temperature interval (12 h, 6 h, 3 h) After filtration with reflux, the filtrate was filtered through a filter paper (Wattmann, USA) and filtered. The filtrate was concentrated under reduced pressure to remove water and ethanol 3: 7 solvent at 40 ° C in a vacuum rotary evaporator. 25 g of 70% (25% yield). Two grams of the sample was used as a sample for antioxidant activity, PTP1B, a-glucosidase and human recombinant aldose reductase inhibitory activity.

< Experimental Example  1>

Tuna heart 70% Ethanol Crude extract DPPH Radical  Measurement of scavenging activity

Blois (1958) used a method to measure the DPPH radical scavenging activity of 70% ethanolic crude extract from tuna hearts. DPPH has an odd number of electrons and exhibits a strong absorption band at 520 nm. At this time, when reacting with an electron donor such as phenol, which provides hydrogen or electrons, electrons or hydrogen radicals are taken from the donor to generate a phenoxy radical. At this time, the absorption band disappears and becomes a stable molecule. In other words, the donated electrons are irreversibly bound to each other, and the color of purple DPPH gradually decreases in proportion to the number of the donated electrons, and the absorbance decreases. Therefore, the decrease of the absorbance of the reaction liquid changing from purple to yellow is measured Radical scavenging activity can be known.

The scavenging activity of DPPH radicals in each sample was measured by dissolving each sample in MeOH and 160 wells and mixing well with 40 μl of 1.5 × 10 ^-4 M DPPH MeOH solution. As a control, MeOH was used. The reaction mixture was allowed to stand at room temperature for 30 minutes and absorbance was measured at 520 nm with a microplate reader spectrophotometer VERSAmax. The radical scavenging activity was expressed as a percentage, and the measurement value was obtained by using the formula (1) repeatedly three times.

As a result, it was found that the radical scavenging activity was increased as the concentration of DPPH radical scavenging of the 70% ethanol extract of tuna heart was increased according to Experimental Example 1 (see FIG. 1). More specifically, the inhibition rate was 74.1% at the highest concentration of 1,600 μg / mL and was 64.7%, 44.6% at the concentration of 800 μg / mL, 400 μg / mL, 200 μg / mL and 100 μg / %, 26.8% and 9.6%, respectively.

In addition, as shown in Table 1, the IC ₅₀ value of the 70% ethanol extract of tuna heart was 503.06 ± 5.02 ㎍ / mL, indicating DPPH radical scavenging activity. The IC ₅₀ value of the positive control group ascorbic acid was 2.25 ± 0.05 ㎍ / mL.

< Experimental Example  2>

Tuna heart 70% Ethanol Crude extract ABTS Radical  Measurement of scavenging activity

To investigate the ABTS radical scavenging ability of 70% ethanolic crude extract of tuna heart, the experimental method used by Re et al. (1999) was modified. Two reagents were mixed at a ratio of 1: 1 (v / v) of 7 mM ABTS (2,2-azino-bis [3-ethylbenzothiazoline-6-sulfonic acid] -diammonium salt) and 2.45 mM potassium persulfate. And allowed to stand for 12-16 hours to form radicals. The ABTS solution was diluted with phosphated buffer saline (pH 7.4) at 734 nm to be 0.70 ± 0.02. The concentration of each sample was adjusted to 20 μl and ABTS solution (180 μl) were added. After stabilization for 2 min, absorbance at 734 nm And expressed as percent inhibition. The percentage of the radical scavenging activity was calculated by the above formula (1).

The ABTS radical scavenging ability of the 70% ethanol extract of tuna heart was confirmed, and it was confirmed in FIG. 2 that the radical scavenging ability increases as the concentration of the sample increases. The inhibition rate was 58.3% at 1000 ㎍ / mL, which was the highest concentration, and 44.6%, 28.7% and 22.1% at the concentrations of 500 ㎍ / mL, 250 ㎍ / mL and 125 ㎍ / mL, respectively.

As shown in Table 2, the IC ₅₀ value of the 70% ethanol crude extract of tuna heart was 867.79 ± 10.20 ㎍ / mL, indicating the ABTS radical scavenging activity. The IC ₅₀ value of the positive control, Trolox, was 2.80 ± 0.27 ㎍ / mL.

< Experimental Example  3>

Tuna heart 70% Ethanol Crude extract ONOO ^- Radical  Measurement of scavenging activity

The ONOO ^- of the 70% ethanol extract of tuna heart is relatively stable compared to other free radicals, but it is easily converted to peroxynitrous acid (ONOOH) which is easily protonated at physiological pH and has high half - life (1.9 s) It is very short and acts as a precursor of many cytotoxic substances nitrogendioxide, nitronium ion and hydroxyl radical, causing oxidation, nitration, hydroxylation reaction. However, since there is no enzyme system for eliminating ONOO ^- in the cells, it is important to find the scavenging active substance. The ONOO ^- scavenging activity was slightly modified by the method of Kooy et al., 1994 to measure the oxidation of DHR 123.

DHR 123 (5 mM) dissolved in ethanol was stored in stock solution at -80 ° C after being charged with nitrogen. The buffer prepared with 90 mM sodium chloride, 50 mM sodium phosphate, and 5 mM potassium chloride was mixed with DTPA (diethylene tetramine penta-acetic acid) 100 so that the final concentration of DHR 123 was 5. Five minutes after the addition of the sample and authentic ONOO ^- to this working solution, the non - fluorescent DHR 123 is converted to the fluorescent rhodamin 123. The fluorescence spectra were measured at excitation and emission wavelengths of 485 and 530 nm, respectively. The results are shown in terms of DHR 123 oxidation inhibition percentage as compared with the negative control to which no sample was added and using Equation 1.

As a result of measuring the peroxynitrite scavenging activity of the 70% ethanol extract of tuna heart, the radical scavenging ability was increased as the concentration of the sample increased. The inhibition rate of 96.2% at the highest concentration of 1000 μg / mL Respectively, and 90.2%, 76.5%, 54.7% and 33.9% inhibition rates were obtained at the concentration of 500 μg / mL, 250 μg / mL, 125 μg / mL and 62.5 μg / mL, respectively. As shown in Table 3, the IC ₅₀ value of the tuna heart 70% ethanol crude extract was 119.87 ± 2.71 μg / mL, indicating ONOO ^- radical scavenging activity. The IC ₅₀ value of the positive control group, penicillamine, was 1.02 ± 0.10 μg / mL.

< Experimental Example  4>

Tuna heart 70% Ethanol Crude extract Total phenol  Content measurement

Total phenol content was determined by colorimetric determination by Folin-Denis method. The extract was dissolved in methanol 5 mg / mL solution and 75 F of Folin reagent were mixed and allowed to stand for 5 minutes. After color development, 200 의 of 5% sodium carbonate solution and 700 의 of distilled water were added. After the mixture was allowed to stand for 60 minutes, the absorbance was measured at 765 nm using a UV-vis spectrophotometer. The total phenolic content in the sample was expressed as mg gallic acid equivalents (GAE) / g sample extract weight, using the calibration curve obtained using gallic acid as a standard substance to measure the total phenolic content.

The total phenol content of 70% ethanol extract of tuna heart was measured by the principle that molybdenum was developed to blue by the reduction of Folin-Ciocalteu regent by the phenolic compound of the extract. To a standard concentration of gallic acid using 0.03125 ~ 0.5 mg / mL concentration was y = 2.5078x + 0.0252 obtain the regression equation (r ² = 0.9998) has a content of 15.80 ± 0.11 GAE mg / g. Fig. 4 shows the results of the gallic acid calibration curve.

< Experimental Example  5>

Tuna heart 70% Ethanol Crude extract PTP1B  Measure inhibitory activity

The method of evaluating the inhibitory activity of PTP1B (Protein tyrosine phosphatase 1B) in tuna heart 70% ethanol crude extract was performed by modifying the method of Cui et al., (2006). The PTP1B (human, recombinant) enzyme used in this experiment was purchased from BIOMAL international LP (USA). The activity of the enzyme was measured using p- nitrophenyl phosphate ( p NPP) as a substrate. For the experiment, 10 μl of sample and 0.5 μg of enzyme Was preincubated at 35 ° C for 5 to 10 minutes, and 50 μl of the substrate ( p NPP) was added. After incubation at 35 ° C for 20 minutes, 10 μl of 10M NaOH was added to terminate the reaction. The absorbance at 405 nm was measured by a microplate reader spectrophotometer and the inhibitory activity of PTP1B was calculated using Equation (1).

As shown in FIG. 5, the PTP1B inhibitory activity was increased as the concentration of the sample increased as a result of evaluating the antidiabetic effect of the 70% ethanol extract of the tuna heart. PTP1B inhibition rates were 85.4%, 68.9%, 53.1%, 40.0% and 16.4% at the concentrations of 2,000 ㎍ / mL, 1,000 ㎍ / mL, 500 ㎍ / mL, 250 ㎍ / mL and 125 ㎍ / mL, .

As shown in Table 4, the IC ₅₀ value of the tuna heart 70% ethanol crude extract was 416.95 ± 4.04 ㎍ / mL, indicating PTP1B inhibitory activity. The IC ₅₀ value of the positive control group, uricolic acid, was 3.88 ± 0.40 ㎍ / mL.

< Experimental Example  6>

Tuna heart 70% Ethanol Crude extract  a- Glucosidase  Measure inhibitory activity

The method of Li et al., 2005 was modified to measure the a-glucosidase inhibitory activity of 70% ethanol extract of tuna heart. To each well, 20 μl of 100 mM phosphate buffer (pH 6.8) and 20 μl of various concentrations of the sample dissolved in 10% DMSO were added. After adding 20 μl of 2.5 mM p- nitrophenyl aD-glucopyranoside ( p NPG) dissolved in 100 mM phosphate buffer (pH 6.8) used as a substrate, 0.2 unit / mL a-glucosidase as an enzyme was added to 10 mM phosphate buffer (pH 6.8) And 20 μl was added. After incubation at 37 ° C for 15 minutes, 80 μl of 0.2 M sodium carbonate solution was added to terminate the reaction. Absorbance was measured at 405 nm using a microplate reader spectrophotometer (Molecular Devices, VERSAmax, CA, USA) and acarbose was used as a positive control. The inhibitory activity of a-glucosidase was calculated using the equation (1) and converted into the IC ₅₀ value.

As a result of evaluating the inhibitory activity of a-glucosidase for confirming the antidiabetic effect of the 70% ethanol extract of tuna heart, it was confirmed that the a-glucosidase inhibitory activity was increased as the concentration of the sample increased as shown in FIG. 5 . , 82.6%, 65.1%, 43.6%, 24.6%, and 8.2% at concentrations of 2,000 ㎍ / mL, 1,500 ㎍ / mL, 1,000 ㎍ / mL, 500 ㎍ / mL, 250 ㎍ / mL and 125 ㎍ / And a-glucosidase inhibition rate of 6.4%. As shown in Table 5, the IC ₅₀ value of the tuna heart 70% ethanol crude extract was 1,136.28 ± 9.36 ㎍ / mL, indicating an a-glucosidase inhibitory activity. The IC ₅₀ value of the positive control group, acarbose, was 75.42 ± 4.82 ㎍ / mL.

< Experimental Example  7>

Tuna heart 70% Ethanol Crude extract  Human recombination Aldoz  Reducing enzyme HRAR ) Inhibitory activity measurement

The method used by Nishimura et al., 1991 was modified to measure the human recombinant aldose reductase inhibitory activity of 70% ethanol extract of tuna heart. To the 1.5-ml quartz cuvette, add 785 μl of 100 mM potassium phosphate buffer (pH 6.2), 5 μl of enzyme, 100 μl of NADPH (0.15 mM) as a coenzyme and 10 μl of the sample dissolved in DMSO. Finally, the substrate, DL-glyceraldehyde Was added to the reaction mixture to make the total reaction volume to 1000 μl, and the spectrophotometer was measured at 340 nm for 1 minute to measure the absorbance reduction rate of NADPH. The control group was measured by adding DMSO instead of the sample, and the positive control group was measured for each concentration using quercetin. The% inhibition activity of human recombinant aldose reductase was determined by the above equation (1) and statistical treatment was performed using linear regression equations.

The human recombinant aldose reductase inhibitory activity of the 70% ethanol extract of tuna heart was measured according to the above method. As shown in FIG. 7, the human recombinant aldose reductase inhibitory activity was found to increase as the concentration of the sample increased Respectively. The inhibition rates were 51.1%, 37.3%, 34.7%, 13.4% and 7.4% at the concentrations of 1000 ㎍ / ml, 800 ㎍ / ml, 400 ㎍ / ml, 100 ㎍ / ml and 50 ㎍ / . As shown in Table 6, the IC ₅₀ value of the tuna heart 70% ethanol crude extract was 984.06 ± 6.08 ㎍ / mL, indicating human recombinant aldose reductase inhibitory activity. The IC ₅₀ value of the positive control, quercetin, was 1.06 ± 0.98 ㎍ / mL.

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 (8)

A pharmaceutical composition for preventing or treating diabetic or diabetic complications comprising tuna heart ethanol extract as an active ingredient.
delete The method according to claim 1,
Wherein the diabetic complication is diabetic retinopathy, diabetic cataract, diabetic neuropathy, diabetic neuropathy, diabetic vascular complication. The method according to claim 1, wherein the tuna heart ethanol extract
(a) placing a tuna heart in an ethanol solvent;
(b) extracting the tuna heart prepared in the step (a) and filtering the filtered tuna; And
(c) removing the ethanol solvent while concentrating the vacuum filtered tuna heart with a vacuum rotary evaporator. 5. The method of claim 4,
Wherein the ethanol solvent of step (a) is water and ethanol mixed in a volume ratio of 3: 7. 5. The method of claim 4,
Wherein the extraction of step (b) is repeated three times at a temperature of 60 to 80 캜 for 1 to 15 hours.
delete Functional health food for prevention or improvement of diabetic or diabetic complication which contains tuna heart ethanol extract as active ingredient.

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