KR101257329B1 - Composition for treating or preventing obesity containing stichpus japonicus extract - Google Patents

Abstract

The present invention relates to a composition for treating or preventing obesity comprising a sea cucumber extract as an active ingredient. In particular, the sea cucumber extract of the active ingredient of the present invention inhibits the differentiation of fat precursor cells (3T3-L1), has the ability to inhibit the proliferation of fat cells, inhibits the accumulation of fat metabolites and is excellent in the treatment and prevention of obesity. In addition, there is no cytotoxicity, and the natural substance as an active ingredient does not cause a problem of side effects can be widely used to treat or prevent obesity.

Description

COMPOSITION FOR TREATING OR PREVENTING OBESITY CONTAINING STICHPUS JAPONICUS EXTRACT}

The present invention relates to a composition for treating or preventing obesity.

Various lifestyle-related diseases and chronic degenerative diseases due to nutritional overcrowding, environmental pollution, lack of exercise, increased stress due to the improvement of economic level are becoming big problems in our society. Of these, obesity is the disease most recently focused on.

The obesity is that energy intake exceeds energy expenditure, and surplus energy causes quantitative and numerical increase of adipocytes, resulting in accumulation of fat tissue or genetic factors, environmental factors. It refers to a disease caused by a breakdown of energy balance in the human body due to factors, mental factors, or lifestyle habits such as eating habits and lack of exercise.

The World Health Organization (WHO) has already recognized it as a disease to treat obesity and has treated it as a global nutrition problem. Currently, the number of obese patients is on the rise, and the World Health Organization recently reported that over one billion adults worldwide are overweight, and at least 300 million of them are obese. According to the 2007 National Health and Nutrition Examination Survey data released by the Ministry of Health and Welfare, the obesity population with a body mass index (BMI) of 25 or more steadily increases and the public health is seriously threatened (Ministry of Health and Welfare (2007 )).

Obesity is particularly prevalent in morbidity and mortality compared to other diseases and has been found to be associated with various complications of adult diseases. For example, obesity patients have been reported to have a higher mortality rate compared to those with normal weight, twice as much as mild illness, 1.6 times as for cerebrovascular disease and 1.8 times as for coronary artery disease. Currently, there are various attempts to treat the diet, including exercise, exercise therapy and diuretics, diarrhea, etc., but its use is temporarily and limitedly limited due to the occurrence of side effects such as nutritional imbalance, decreased immunity and depression.

Regarding the above-mentioned obesity, studies on localization (Adipogensis) are under way. The localization refers to a process in which adipocytes are differentiated from fat precursor cells to accumulate fat, and the localization is known to cause metabolic diseases such as obesity, diabetes, fatty liver and coronary heart disease. The adipocyte functions not only as an energy reservoir but also as an endocrine organs, which secretes a protein hormone called adipocytokine to perform various functions for maintaining energy homeostasis. However, excessive adipocyte differentiation and unbalanced energy metabolism may cause abnormal gene expression and signal transduction in adipocytes, leading to adipocytokine secretion abnormalities.

Leptin, adiponectin, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and resistin have been reported as the types of the adipocyte kine. For example, leptin, an appetite regulating protein, decreases appetite and increases energy expenditure, and the adiponectin increases expression during adipocyte differentiation, thereby increasing liver and muscle tissue insulin sensitivity, increasing fatty acid oxidation, Or not functioning, obesity may be induced, and it has been reported that the decrease in the expression level of adiponectin and the induction of obesity are related to each other.

In fat cells, lipid metabolism occurs through lipolysis and adiogenesis, and during the fat synthesis, the preadipocytes are differentiated into mature adipocytes through proliferation and differentiation. This forms a lipid droplet in the cell. Transcription factors that induce differentiation of adipocytes include adipocyte determination and differentiation dependent factor 1 / sterol response element binding protein 1c (ADD1 / SPEBP1c), peroxisome proliferator activated receptor γ (PPARγ), and C / EBPα (CCAAT). enhancer binding protein α) has been reported. The transcription factors are induced at different time points during differentiation of adipocytes, and they interact with each other to regulate the expression of adipocyte-specific genes, gradually inducing lipid metabolism and adipocyte differentiation.

The mechanisms of obesity treatment for eliminating obesity and maintaining proper body weight include controlling appetite, preventing digestion and absorption of fat, increasing energy consumption, and controlling lipid metabolism. Currently used anti-obesity drugs include drugs that inhibit the activity of serotonin and lipolytic enzymes, which interfere with the hydrolysis of cholesterol and triglycerides and help excrete unoxidized fats into stools. Orlistat (XenicalR).

However, sibutramine should be used with caution in patients with cardiovascular diseases by increasing blood pressure, and orlistat has been reported to induce digestive problems, fatty stools, bowel incontinence, and fat-soluble vitamin absorption.

Therefore, the development of anti-obesity drugs that have high anti-obesity effects and low side effects among natural products that are expected to minimize the possibility of occurrence of safety problems are required. In other words, while inhibiting the differentiation process of the adipocytes, the development of a weaker natural-derived material is required.

Patent application No. 2007-0060222 Patent Application No. 2007-0060221 Patent Application No. 2007-0056226 Patent Application No. 2006-0044559 Patent application No. 2000-0021902

The present invention is to solve the problems of the prior art as described above, it is an object of the present invention to provide a composition for the treatment or prevention of obesity comprising a sea cucumber extract as an active ingredient.

The present invention relates to an anti-obesity composition comprising sea cucumber extract as an active ingredient in order to achieve the above object.

The present invention also relates to a composition for treating or preventing obesity, comprising a sea cucumber extract as an active ingredient in order to achieve the above object. The sea cucumber may be Stichopus japonicus Selenka. The sea cucumber extract may be a hot water extract of the sea cucumber gut.

In addition, the present invention relates to a food composition for preventing or improving obesity comprising sea cucumber extract as an active ingredient in order to achieve the above object. Examples of the food composition of the present invention include food, food additives, beverages or beverage additives.

In addition, the present invention by cutting the sea cucumber guts ( Stichopus japonicus Selenka), separating the guts sea cucumber guts and adding water to the guts of the separated protrusion sea cucumbers and extracted for 1 to 5 hours at 200 ℃ to 300 ℃ It relates to a method for producing sea cucumber gut extract having an anti-obesity and weight loss effect, including a hot water extraction step.

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

The inventors of the present invention, while studying a natural substance-derived adipocyte differentiation inhibiting material with fewer side effects and easy to administer, as a result of confirming using 3T3-L1 cells, mouse-derived progenitor cells, sea cucumber extract, in particular sea cucumber guts It was confirmed that the extract of S. erythematosus was effective in inhibiting the production of adipocytes, that is, in the differentiation of adipocytes, and in inhibiting the adipogenesis of adipocytes. After confirming this, the present invention was completed.

The present invention relates to an anti-obesity composition comprising sea cucumber extract as an active ingredient in order to achieve the above object. The anti-obesity composition may be a pharmaceutical composition for treating or preventing obesity or a food composition for improving or preventing obesity. The anti-obesity composition may be a composition having an anti-obesity effect and a weight loss effect.

The present invention also relates to a composition for treating or preventing obesity, comprising a sea cucumber extract as an active ingredient in order to achieve the above object. The composition for treating or preventing obesity may be a pharmaceutical composition for treating or preventing obesity. The sea cucumber is Stichopus japonicus Selenka). The sea cucumber extract may be a hot water extract of the sea cucumber gut.

In the present invention, the sea cucumber (sea cucumber) are echinoderms dongmulmun (Phylum Echinodermata) means a general term of echinoderms belonging to sea cucumber steel (Class Holothuroidea), and the sea cucumber projection sea cucumber (Stichopus japonicus Selenka belonging to sea cucumber steel pure neck (Order Aspidochirotida), jasam and or projections sea cucumber and (Family Stichopodidae)), spiny sea cucumber (Stichopus horrens Selenka), blue sea cucumber ( Stichopus) chloronotus Brandt), including the echinoderm belonging to Stichopus variegatus Semper, preferably may be Stichopus japonicus Selenka widely distributed in the waters of Korea, Japan and China.

The sea cucumber is also referred to as Moksam, Namako, seaman, meat, sea bass, sonson, meat, seaman or blackworm.

The sea cucumber is a male and female body, in vitro fertilization, the body is a long cylindrical shape back and forth, there are a number of bumps on the back. The anterior end of the sea cucumber's body has an open mouth, several tentacles around it, and an anterior end. In addition, most sea cucumbers have many coffins, which are made of coffins on the bottom, which crawl under the sea. Species without irrigation among the sea cucumbers live in the sea or buried in sand mud. The skin of the sea cucumber contains sparse small bone fragments.

The sea cucumber is vigorous appetite and active in the water temperature below 17 ℃, stop eating when the temperature is above 17 ℃, sleep over 25 ℃ summer sleep. When the sea cucumbers are eaten, they put sand mud under the sea with their tentacles in their mouths to eat small creatures in the sand mud, and sand and dung are sent out. When the sea cucumber is stimulated from the outside, it breaks the intestine and sends it out of the anus.

The sea cucumber extract may be prepared by adding a fractional solvent to the crude extract prepared by extracting or extracting with an extraction solvent.

The extraction solvent may be at least one selected from the group consisting of water and an organic solvent. The organic solvent may be a polar solvent such as methanol, ethanol or the like, an alcohol having 1 to 5 carbon atoms, ethyl acetate or acetone, or a nonpolar solvent such as ether, chloroform, benzene, hexane or dichloromethane or a mixture thereof. The alcohol having 1 to 5 carbon atoms may be an alcohol or an aqueous alcohol solution, preferably an aqueous solution of 1 to 5 carbon atoms such as 50 to 99% ethanol, and more preferably 50 to 95% carbon atoms such as ethanol It may be 1 to 5 aqueous alcohol solution.

The extract of the present invention may be prepared according to a method for preparing an extract for a target animal, specifically a mollusc, and specifically, may be cold extraction, hot extraction or heat extraction, etc. Ultrasonic grinding extractors or fractionators can be used.

In addition, the extract of the present invention may be a fraction further subjected to the fractionation process after adding a fractional solvent to the crude extract extracted with the solvent. The fractional solvent may be a solvent selected from the group consisting of water, ethyl acetate, ether, chloroform, benzene, hexane, methylene chloride and mixed solvents thereof.

The thus-prepared extract or the fraction obtained by performing the fractionation process may be filtered, concentrated or dried to remove the solvent, and may be subjected to both filtration, concentration and drying. Specifically, the filtration can be performed using a filter paper or a vacuum filter, and the concentration can be reduced by using a vacuum concentrator, for example, a rotary evaporator. The drying can be performed by, for example, freeze drying.

In one embodiment of the present invention, the sea cucumber extract may be obtained by the following method.

The sea cucumber extract includes cutting the sea cucumber, specifically, the protrusion sea cucumber, the sea cucumber gut separation step for separating the guts and the extraction step of adding water to the separated guts and extracting for 1 hour to 5 hours at 200 ℃ to 300 ℃ The extracting step of extracting may further include a step of concentrating the extract solution under reduced pressure and / or a lyophilization step of freezing and drying the extract or concentrated under reduced pressure.

Sea cucumber extract which is an active ingredient of the composition for treating or preventing obesity is preferably an extract of Stichopus japonicus Selenka, more preferably, a hydrothermal extract of the intestines of the processus sea cucumber, specifically water in the intestine separated from the processus sea cucumber After adding, may be extracted by applying heat.

In the present invention, obesity (obesity) is a genetic factor, an environmental factor, a mental factor, or the energy balance in the human body is disrupted by lifestyle, such as eating habits and lack of exercise, that is, energy intake (energy intake) energy consumption (energy expenditure) ) Surplus energy causes quantitative and numerical increase of adipocytes, resulting in the accumulation of adipose tissue. Obesity is particularly prevalent in morbidity and mortality compared to other diseases and has been found to be associated with various complications of adult diseases.

Obesity treatment or prevention composition comprising the sea cucumber extract of the present invention as an active ingredient to include the active ingredient, specifically the sea cucumber extract 0.001 to 99.99% by weight, preferably 0.1 to 99% by weight relative to the total weight of the composition Can be.

Obesity treatment or prevention composition comprising the sea cucumber extract, specifically the sea cucumber gut hot water extract as an active ingredient does not cause problems of side effects in that it is an extract of natural substances, there is no cytotoxicity as a result of the improvement of obesity or It has an excellent effect in that it has an excellent preventive effect.

Obesity treatment or prevention composition comprising the sea cucumber extract of the present invention as an active ingredient can be applied directly to humans.

The composition for treating or preventing obesity comprising the sea cucumber extract as an active ingredient may include the sea cucumber extract as an active ingredient alone or further include a substance having an anti-obesity effect on the sea cucumber extract as an active ingredient, and other formulations, Depending on the method of use and purpose of use, it may further comprise additional ingredients, pharmaceutically acceptable or nutritionally acceptable carriers, excipients, diluents or accessory ingredients.

More specifically, the composition for treating or preventing obesity comprising the sea cucumber extract as an active ingredient, in addition to the active ingredient, nutritional supplements, vitamins, electrolytes, flavors, coloring agents, neutralizing agents, pectic acid and salts thereof, alginic acid and salts thereof , Organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. In addition, the carrier, excipient or diluent may be lactose, dextrose, sucrose, sorbitol, mannitol, ziitol, erythritol, maltitol, starch, acycia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline In the group consisting of cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, dextrin, calcium carbonate, propylene glycol, liquid paraffin, saline It may be one or more selected, but is not limited to any conventional carrier, excipient or diluent can be used. The components may be added independently or in combination with the active ingredient, that is, sea cucumber extract.

Obesity treatment or prevention composition comprising the sea cucumber extract as an active ingredient is 0.001% to 99.9% by weight, preferably 0.1% to 99% by weight, more preferably 1% by weight of the active ingredient relative to the total weight of the composition % To 50% by weight. In addition, the content of the additional component is preferably added in the range of 0.1 to 20 parts by weight per 100 parts by weight of the composition for treating or preventing obesity.

In addition, when pharmaceutical compositions for treating or preventing obesity containing the sea cucumber extract as an active ingredient, conventional fillers, extenders, binders, disintegrants, surfactants, anti-coagulants, lubricants, wetting agents, flavors, emulsifiers or preservatives, etc. It may further include, can be used both oral or parenteral.

Specifically, the solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations may include at least one excipient such as starch, calcium carbonate (calcium) in the active ingredient, that is, sea cucumber extract. carbonate, sucrose or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral use include suspensions, solvents, emulsions, and syrups.In addition to the commonly used simple diluents, water and liquid paraffin, various excipients may include, for example, wetting agents, sweeteners, fragrances, and preservatives. have.

In addition, the formulation of the composition for the treatment or prevention of obesity comprising the sea cucumber extract of the present invention as an active ingredient may be in a preferred form according to the method of use, and in particular, provides a rapid, sustained or delayed release of the active ingredient after administration to a mammal. It can be formulated by adopting a method known in the art to do so. Examples of the specific formulations include granules, powders, syrups, liquids, suspensions, tablets, injections, main tablets, cataplasma, capsules, soft or hard gelatin capsules and the like.

Furthermore, a composition for treating or preventing obesity comprising the sea cucumber extract of the present invention as an active ingredient can be prepared using Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Science, recent edition), Mack Publishing Company, Easton PA Formulated preferably using the method disclosed herein.

The dosage of the composition for treating or preventing obesity comprising the sea cucumber extract according to the present invention as an active ingredient may be appropriately selected by those skilled in the art in consideration of the method of administration, the age, sex and weight of the recipient, and the severity of the disease. have. For example, the composition for treating or preventing obesity comprising the sea cucumber extract of the present invention as an active ingredient, based on the active ingredient, 0.0001 mg / kg to 1000 mg / kg, in order to be more effective from 0.01 mg / kg to It may be administered at 100 mg / kg. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

In addition, the composition for treating or preventing obesity comprising the sea cucumber extract of the present invention as an active ingredient, in addition to the active ingredient may further include an extract for a compound or natural product having a known obesity treatment or prevention effect, the active ingredient 5 parts by weight to 200 parts by weight with respect to 100 parts by weight, respectively.

In addition, the present invention relates to a food composition for preventing or improving obesity comprising sea cucumber extract as an active ingredient in order to achieve the above object.

The sea cucumber may be Stichopus japonicus Selenka. The sea cucumber extract may be a hot water extract of the sea cucumber gut. The hydrothermal extract of the sea cucumber gut is a sea cucumber ( Stichopus japonicus Selenka) by cutting the sea cucumber gut separation step to separate the guts and hot water extraction step of adding water to the guts of the separated protruding sea cucumber and extracted for 1 hour to 5 hours at 200 ℃ to 300 ℃ Can be prepared.

As used herein, the term " food " means a natural product or a processed product containing one or more nutrients. Preferably, it means that the food can be directly eaten through a certain degree of processing. Health functional foods, beverages, food additives, and beverage additives.

The food of the present invention includes, for example, various foods, beverages, gums, tea, a vitamin complex, a health functional food, and the like. In addition, the food in the present invention includes special nutritional products (e.g., prepared oils, infants, baby food, etc.), processed meat products, fish products, tofu, jelly, noodles (e.g. ramen, noodles, etc.), health supplements, seasoned foods ( For example, soy sauce, miso, red pepper paste, mixed soy sauce), sauces, confectionery (e.g. snacks), dairy products (e.g. fermented milk, cheese, etc.), other processed foods, kimchi, pickles (various kimchi, pickles, etc.), beverages ( Examples include, but are not limited to, fruits, vegetable drinks, soy milk, fermented beverages, and the like, and natural seasonings (eg, ramen soup). The food, the health functional food, the beverage, the food additive and the beverage additive can be produced by a usual production method.

In the present invention, a health functional food is a biological defense rhythm control, disease prevention and the like having a food group or a food composition which has added value to the food by using physical, biochemical, biotechnological techniques, etc. It refers to a food that is designed and processed to fully express the weight control function related to recovery.

The health 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 health functional foods.

In the present invention, beverage is a generic term for drinking or enjoying a taste, and is intended to include a health functional beverage. The beverage is not particularly limited to other ingredients other than including the sea cucumber extract as an active ingredient in the indicated ratio as an active ingredient, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. .

Examples of such natural carbohydrates include monosaccharides such as glucose, fructose and other disaccharides such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin and the like, and Sugar alcohols such as xylitol, sorbitol, and erythritol. Natural flavors (tau martin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate may be generally about 1 to 20 g, preferably 5 to 12 g per 100 ml of the food composition of the present invention. In addition, the composition of the present invention can be used for the production of natural fruit juice, fruit juice drink, Can also be added.

In addition to the above, the food composition of the present invention can be used as a flavoring agent such as a variety of nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and thickening agents (cheese, chocolate etc.), pectic acid and its salts, Salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. These components can be used independently or in combination. The proportion of such additives is not so critical, but may be selected in the range of 0 to 2,000 parts by weight per 100 parts by weight of the sea cucumber extract of the present invention.

In the present invention, the health functional beverage is a biological defense rhythm control or disease prevention of a beverage group or beverage composition which has added value to the beverage by using physical, biochemical and biotechnological techniques to act and express the function of the beverage for a specific purpose. Means a beverage that is designed and processed to fully express the gymnastic function related to recovery and the like.

The health functional beverage is not particularly limited to other ingredients except the sea cucumber extract of the present invention as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks.

Examples of such natural carbohydrates include monosaccharides such as glucose, fructose and other disaccharides such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin and the like, and xylitol Sugar alcohols such as sorbitol and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of natural carbohydrates is generally about 1 to 20 g, preferably 5 to 12 g per 100 ml of the composition of the present invention.

In addition, in the food composition for preventing or improving obesity containing sea cucumber extract as an active ingredient, the active ingredient may comprise 0.01 to 15% by weight of the total food weight, the beverage composition is 0.02 to 5 based on 100 ml g, preferably 0.3 to 1 g.

The sea cucumber extract of the present invention has been confirmed that the anti-obesity effect, in particular, inhibiting the effect of adipocyte differentiation and adipocyte size increase, the composition comprising the sea cucumber extract of the present invention as an active ingredient has excellent anti-obesity prevention or improvement effect It was confirmed to be visible.

The present invention also relates to an anti-obesity composition having an anti-obesity and weight loss effect or a method for producing a sea cucumber viscera extract which is an active ingredient of the anti-obesity composition.

Specifically, the manufacturing method may include a sea cucumber hot water extraction step of adding water and heat to the guts of sea cucumbers, preferably dolgis ( Stichopus japonicus Selenka), more preferably dolgis.

The production method is a sea cucumber, preferably the process of cutting the sea cucumber, sea cucumber gut separation step for separating the guts and water is added to the guts of the separated protrusion sea cucumber and 200 ℃ to 300 ℃, preferably 230 ℃ to 270 ℃ It relates to a method for preparing sea cucumber viscera extract having an anti-obesity and weight loss effect comprising a hot water extraction step for 1 hour to 5 hours, preferably 2 hours to 4 hours.

The manufacturing method may further include a step of concentrating the hot water extract under reduced pressure and / or a freeze drying step of freezing and drying the concentrated water concentrate of the hot water extract or the hot water extract.

As described above, an anti-obesity composition comprising the sea cucumber extract of the present invention as an active ingredient, for example, a composition for treating or preventing obesity comprising the sea cucumber extract as an active ingredient or for preventing or improving obesity comprising the sea cucumber extract as an active ingredient Ingestion of the food composition or sea cucumber extract which is an active ingredient of the composition inhibits the differentiation into adipocytes and inhibits the increase of adipocyte size, which has been confirmed to have an anti-obesity or weight loss effect.

Since the sea cucumber gut hot water extract of the present invention or the composition of the present invention has the effect of inhibiting the differentiation of fat cells and the growth inhibitory effect of adipocytes, it is effective in weight loss and obesity treatment, improvement or prevention, as well as existing synthetic compounds Unlike the therapeutic agent for obesity, the active ingredient of the present invention is an extract of natural products does not have any side effects, and there is no cytotoxicity, and furthermore, since it is used as an active ingredient in sea cucumbers inhabiting abundantly in the coast of Korea, it is also economically positive, The composition will be industrially very effective.

1 is a graph measuring the survival rate of the fat precursor cells (3T3-L1) when the sea cucumber extract treatment in order to confirm the toxicity of the sea cucumber extract according to an embodiment of the present invention.
Figure 2 is a graph confirming the proliferation inhibitory ability of the differentiation cells of the sea cucumber extract according to an embodiment of the present invention, the horizontal axis shows the type of the control (Control) and the sample, the vertical axis shows the fat producing ability, Nor is fat Progenitor cells, PC for adipocytes, Sjh for sea cucumber hydrothermal extract, Sjh-h for hexane fraction of sea cucumber hydrothermal extract, Sjh-c for chloroform fraction for sea cucumber hydrothermal extract, Sjh-e for ethyl acetate fraction of sea cucumber hydrothermal extract, Sjh- b is a butanol fraction of sea cucumber hydrothermal extract, Sjh-w is a water fraction of sea cucumber hydrothermal extract, Sjv is a hexane fraction of sea cucumber gut hydrothermal extract, and Sjh-h is a hexane fraction of sea cucumber hydrothermal extract.
Figure 3 is a photograph confirming the proliferation inhibitory ability of the differentiation cells of the sea cucumber extract according to an embodiment of the present invention, Nor is fat progenitor cells, PC is fat cells, Sjh is the sea cucumber hydrothermal extract, Sjh-h of sea cucumber hydrothermal extract Hexane fraction, Sjh-c is the chloroform fraction of sea cucumber hydrothermal extract, Sjh-e is the ethyl acetate fraction of sea cucumber hydrothermal extract, Sjh-b is the butanol fraction of sea cucumber hydrothermal extract, Sjh-w is the water fraction of sea cucumber hydrothermal extract, Sjv is Hexane fraction of sea cucumber viscera hydrothermal extract, Sjh-h represents the hexane fraction of sea cucumber hydrothermal extract.

Hereinafter, the structure and effects of the present invention will be described in more detail with reference to specific examples and comparative examples in order to facilitate understanding of the present invention. It should be understood, however, that the following Examples are for the purpose of more clearly understanding the present invention and that the scope of the present invention is not limited by the following Examples, but should be construed in accordance with the appended claims and all equivalents thereof The technical idea is to be construed as falling within the scope of the present invention.

[Example]

Example 1: Preparation of Extract

250 g of sea cucumbers collected from the coast of Haenam-gun, Jeollanam-do, Korea were obtained and washed in running water to remove foreign substances, and then the internal organs were separated. Extracts were prepared from the intestines of sea cucumbers and sea cucumbers, each of which was isolated.

More specifically, in the case of sea cucumber extract, after cutting the sea cucumber separated the internal organs at intervals of 100 mm, 40 g of the cut sea cucumber is placed in an extraction container, 1 L of distilled water is added, and reflux extraction at 250 ° C. for 3 hours. It was prepared by the method. After the prepared extract was filtered under reduced pressure using a vacuum filter, the filtered extract was concentrated using a vacuum condenser on a water bath at 50 ° C., and then frozen in a freeze dryer system at −70 ° C. for 24 hours. Dried sea cucumber hot water extract was prepared.

Fractions of sea cucumber hot water extract was put into the extraction vessel 40 g of the sea cucumber cut at 100 mm intervals, 1 L of distilled water was added and then refluxed at 250 ℃ for 3 hours. The prepared extract was added to the fractional filter, and 1 L of hexane (Hexane) was added, mixed and fractionated to separate the hexane layer and the aqueous layer, which was an hexane-insoluble layer, and then hexane fraction was prepared by obtaining only the hexane layer. 1 L of chloroform was added to the hexane insoluble layer (aqueous layer), followed by mixing and fractionation to separate the chloroform layer and the chloroform insoluble layer (aqueous layer), and then obtaining only a chloroform layer to prepare a chloroform fraction. Ethyl acetate 1L was added to the chloroform insoluble layer (aqueous layer), followed by mixing and fractionation to separate the ethyl acetate layer and the ethyl acetate insoluble layer (aqueous layer), thereby obtaining ethyl acetate fraction to prepare an ethyl acetate fraction. Butanol 1L was added to the ethyl acetate insoluble layer (aqueous layer), followed by mixing and fractionation to separate the butanol layer and the butanol insoluble layer (aqueous layer), and obtaining only butanol layer, thereby preparing a butanol fraction. Each of the obtained fractions was concentrated by filtration through a vacuum filter, and then lyophilized at -20 ° C to completely remove the solvent, and then used in this experiment.

In the case of sea cucumber gut extract, 40 g of the sea cucumber gut was placed in an extraction container, and 1L of distilled water was added thereto, followed by reflux extraction at 250 ° C. for 3 hours to prepare a sea cucumber gut extract. After the prepared extract was filtered under reduced pressure using a vacuum filter, the filtered extract was concentrated using a vacuum condenser on a water bath at 50 ° C., and then frozen in a freeze dryer system at −70 ° C. for 24 hours. Dried sea cucumber gut hot water extract was prepared. The obtained sea cucumber viscera extract was concentrated by filtration through a vacuum filter, and then freeze-dried at -20 ° C to completely remove the solvent, and then used in this experiment.

Example 2: Cytotoxicity Test (MTT assay) of Sea Cucumber Extract

In order to measure the cytotoxicity of the sea cucumber extract, fraction and sea cucumber gut extract prepared in Example 1, mouse-derived 3T3-L1 adipocytes were purchased from ATCC.

The mouse fat precursor cells 3T3-L1 were dispensed with a 96-well plate containing 10% BCS DMEM medium, and the cells were cultured at 37 ° C. and wet CO (5% CO 2/95% air). The cells were re-dispensed at a density of 50,000 cells / well in a 24 well plate and maintained for 2 more days at the 100% confluency time point.

Adipose progenitor cells induced differentiation by fat tax for 3 days with 10% FBS DMEM medium containing MDI (0.5 mM 3-iso-butyl-methyxathine (IBMX), 1 uM dexamethasone, 1 ug / ml insulin). Incubated for 3 days with 10% FBS DMEM culture containing ug / ml insulin. All 12 days of differentiation confirmed that about 80% or more of the cells were differentiated.

After differentiation, the sea cucumber hot water extract, the hot water extract fraction, and the sea cucumber intestine extract were divided into experimental groups, which were further cultured for 24 hours, and then the culture solution was removed and the number of living cells was measured by MTT assay.

The MTT analysis was performed in the following manner. First, after removing the cell culture medium, 1 ml of DMEM / F12 medium containing 1 mg / ml of MTT [3- (4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide] was treated 1 ml per well. And further incubated for 4 hours in a CO 2 incubator at 37 ° C. After removing the medium, the tetrazolium bromide salt was removed, and 200 μl of DMSO was dissolved to dissolve the formazan crystal generated in each well, and then the absorbance was measured at 540 nm with a microplate reader (BIO-RAD) to measure the cells. The survival rate was confirmed. The results are shown in FIG.

As shown in FIG. 1, the sea cucumber hot water extract prepared in Example 1 and the hot water extract fraction and various concentrations of the visceral extract, in particular, the concentration of 10 ug / ml to 200 ug / ml as a result of the treatment, the extract Both and fractions were found to show little effect on the proliferation of the cells. Therefore, it was confirmed from the results that the sea cucumber extract, fraction and sea cucumber gut extract are all cytotoxic up to 200 ug / ml.

Example 3: Anti-obesity effect measurement of sea cucumber extract

The anti-obesity effect was measured using sea cucumber extract, fraction and sea cucumber viscera extract 200 ug / ml and 3T3-L1 adipocytes, which were confirmed to be non-cytotoxic in Example 2.

More specifically, the 3T3-L1 adipocytes of Example 2 were cultured in a culture condition of 37 ℃ and wet conditions (5% CO ₂ /95% O ₂ air) using 10% BCS DMEM medium. The cells were re-dispensed at a density of 50,000 cells / well in a 24 well plate and maintained for 2 more days at the 100% confluency time point.

The progenitor cells induced differentiation by fat tax for 10 days with 10% FBS DMEM medium containing MDI (0.5 mM 3-iso-butyl-methyxathine (IBMX), 1 uM dexamethasone, 1 ug / ml insulin), again Incubated for 3 days with 10% FBS DMEM medium containing 1 ug / ml insulin. All 12 days of differentiation confirmed that about 80% or more of the cells were differentiated.

After differentiation, sea cucumber hot water extract, hot water extract fraction, and sea cucumber intestine extract were divided into experimental groups, respectively, and further incubated for 6 hours, washed twice with PBS, and added 4% formaldehyde with 1% (v / v) calcium chloride. The cells were fixed at room temperature for 30 minutes. After the fixed cells were dried, the cells were treated with 4% Oil Red O solution (Oil Red O staining reagent), which was saturated with absolute alcohol and filtered with a 0.4 um filter for 40 minutes, and the Oil Red O staining reagent was purified by PBS. After washing enough times, the stained adipocytes were washed once with 60% isopropanol, and observed in the adipocytes under a microscope to evaluate the degree of adipogenesis, it was shown in Figure 3, the stained adipocytes 60% After eluting with isopropanol, absorbance at 560 nm was measured and shown in FIG. 2.

As shown in FIG. 2 and FIG. 3, it was confirmed that the production of fat droplets in the sample treatment group was significantly reduced, as compared to the control group (PC), which was not treated with the sample. In comparison, sea cucumber visceral hot water extract was found to have about 80% fat accumulation in the control group (PC) without the extract or fractions, which was superior to the sea cucumber viscera extracts in inhibiting fat production. Confirmed.

From the above results, it was confirmed that the sea cucumber viscera extract has an anti-obesity effect and weight gain suppression effect.

Manufacturing example : Preparation using sea cucumber viscera extract

Manufacturing example  One. Sanje  Produce

Sea cucumber viscera extract powder 20 mg

Lactose 100 mg

Talc 10 mg

The above ingredients were mixed and filled in an airtight container to prepare powders.

Manufacturing example  2. Preparation of tablets

Sea cucumber viscera extract powder 10 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets were prepared by tableting according to the usual preparation method of tablets.

Manufacturing example  3. Preparation of Capsule

Sea cucumber viscera extract powder 10 mg

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2 mg

The above components were mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

Manufacturing example  4. Liquid  Produce

Sea cucumber viscera extract powder 20 mg

10 g of isomerized sugar

5 g of mannitol

Purified water quantity

After dissolving each component in purified water according to the usual method of preparing a liquid solution, adding lemon flavor appropriately, mixing the above components, adding purified water, adjusting the whole to 100 ml by adding purified water, and then filling into a brown bottle. The solution was prepared by sterilization.

Manufacturing example  5. Preparation of dietary supplements

Sea cucumber viscera extract powder 1,000 mg

Vitamin mixture proper amount

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

0.13 mg vitamin B1

0.15 mg of vitamin B2

Vitamin B6 0.5 mg

0.2 μg of vitamin B12

10 mg vitamin C

Biotin 10 μg

Nicotinic acid amide 1.7 mg

50 μg folic acid

Calcium pantothenate 0.5 mg

Mineral mixture

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dibasic calcium phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixtures is a composition that is relatively suitable for the health functional food, the composition is mixed in a preferred embodiment, but the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health functional food manufacturing method. The granules can then be prepared and used in the manufacture of nutraceuticals in accordance with conventional methods.

Claims (5)

delete delete delete delete Sea cucumber viscera separation step of cutting the guts ( Stichopus japonicus Selenka), to separate the viscera; And hot water extraction step of adding water to the intestines of the separated protrusion sea cucumber and extracting at 200 ° C. to 300 ° C. for 1 hour to 5 hours.
Method of producing a sea cucumber gut extract having an anti-obesity and weight loss effect comprising a.

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