KR102014413B1 - Composition for anti-obesity comprising extract of Scapharca broughtonii as effective component - Google Patents

Abstract

The present invention relates to an anti-obesity composition comprising extract of Anadara broughtonii as an active ingredient. More particularly, the Anadara broughtonii ethanol extract of the present invention has no cytotoxicity and is excellent in inhibiting the differentiation of adipocytes, and thus the composition of the present invention containing the Anadara broughtonii ethanol extract as an active ingredient can be usefully used as an anti-obesity functional health food or a therapeutic agent for obesity.

Description

Composition for anti-obesity comprising extract of Scapharca broughtonii as effective component

The present invention relates to an anti-obesity composition containing the shellfish extract as an active ingredient.

Obesity is a state in which the excessive accumulation of adipose tissue in the body, in the case of obesity generally loses a lot of weight, but even a large number of muscles even if not obesity, because the excess fat tissue in the body is defined as obesity do. Excessive intake of nutrients relative to energy consumption over long periods of time leads to obesity caused by energy imbalances, as well as a combination of hormonal changes, heredity, mental health problems and socioeconomic factors. As the obesity population is increasing all over the world, it has recently been identified as an important factor causing adult disease, and the severity of obesity has emerged as an important problem.

In order to prevent and improve obesity and maintain a healthy weight, you need to take care of your life through balanced diet and regular exercise. In particular, once the fat cells are made, they can be reduced in size, but they are impossible to remove naturally, so they remain permanently in the human body.

In order to prevent, improve or treat obesity, a method of taking drugs such as reducing calorie intake, increasing activity and exercise amount, and reducing fat absorption are used.

Current treatments for obesity include fenfluramine, which inhibits the serotonin nervous system, ephedrine and caffeine through the noradrenergic nervous system, and sibutramine and pancreatic lipase, which simultaneously act on the serotonin and noradrenaline nervous system, to reduce fat absorption. Drugs such as start. However, among the previously used drugs, fenfluramine has been banned due to side effects such as primary pulmonary hypertension and heart valve lesions, and other drugs are also used for patients with heart failure or kidney disease due to problems such as decreased blood pressure or lactic acidosis. There is a problem that can not be. Therefore, development of a substance for preventing, improving or treating obesity with less side effects is required.

On the other hand, Scapharca broughtonii ) is a shellfish belonging to the tympanic clam, and its shape is ovate, and both clams are well swelled and the thinnest among tympanic. On the surface of clamshell, there are 42 ~ 43 trillion convex radiant 륵 (射 勒 射 勒), and above it is a blackish brown cuticle. The inside of clamshell is white and is a large clam that is 120mm long, 90mm high, and 75mm wide. It is widely distributed in the southern and eastern coasts of Korea, and it lives from the low tide line to the depth of 50m and lives mostly in the low quality sand soil.

The shellfish are oviparous, and eggs laid in summer are fertilized in seawater and then floated for two to three weeks. The duration of attachment life is about 2 months and depends on the environment of the place of attachment. After a period of attachment, they sneak into inferior quality and live.

Shellfish are eaten raw, but recently they have started to be used as raw materials for canning. Total shellfish has total calories per 100g, 81㎈, 79.8g water, 15.5g protein, 0.5g fat, 3.5g carbohydrate and 0.7g ash, and other vitamins A, B1, B2, C and niacin It is known to be effective in preventing anemia, enhancing digestive function, protecting eyesight, relieving fatigue, detoxifying liver and maintaining bone density.

Meanwhile, Korean Patent No. 1673515 discloses a health food composition for improving obesity using a tympanum membrane, and Korean Patent No. 1847452 discloses an antihypertensive composition containing an extract of shellfish as an active ingredient, but according to the present invention, There is no disclosure regarding an anti-obesity composition containing the shellfish extract as an active ingredient.

The present invention was derived by the above-mentioned requirements, and completed the present invention by providing a composition for anti-obesity containing the shellfish extract as an active ingredient, and confirming the significant inhibitory effect of differentiation of the shellfish ethanol extract.

In order to solve the above problems, the present invention provides an anti-obesity health functional food composition containing the shellfish extract as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of obesity containing the shellfish extract as an active ingredient.

The present invention relates to an anti-obesity composition containing the shellfish extract as an active ingredient, the shellfish ethanol extract is not cytotoxic, and the effect of inhibiting the differentiation of adipocytes is remarkable, containing the shellfish ethanol extract as an active ingredient The composition of the present invention can be used as an anti-obesity health food composition or a pharmaceutical composition for the prevention or treatment of obesity.

1 is a result confirming the cytotoxicity of the shellfish extract of the present invention. Control is the group that did not process the sample.
Figure 2 is a result confirming the inhibitory effect of adipocyte differentiation by concentration of the shellfish extract of the present invention. Control is a group that did not process the sample when inducing adipocyte differentiation. ** means that the adipocyte differentiation rate of Experimental Example 1 was significantly reduced compared to the hydrothermal extract treatment group of Comparative Example 1, and p <0.01.

The present invention relates to an anti-obesity dietary supplement composition containing the shellfish extract as an active ingredient.

The solvent of the extract may be water, a lower alcohol of C ₁ ~ C ₄ or a mixture thereof, preferably extracted using ethanol as a solvent, but is not limited thereto.

The extract may be used at a concentration of 300 µg / ml or more, preferably 300 µg / ml to 1200 µg / ml, but is not limited thereto.

The shellfish extract is a safe substance without cytotoxicity, and can inhibit the differentiation of fat cells.

The composition may be prepared in any one of a powder, granules, pills, tablets, capsules, candy, syrups and beverages, but is not limited thereto.

When the health functional food composition of the present invention is used as a food additive, the health functional food composition may be added as it is or used with other foods or food ingredients, and may be appropriately used according to a conventional method. The active ingredient may be appropriately used depending on the purpose of use (prevention or improvement). In general, in the preparation of food or beverages, the nutraceutical composition of the present invention is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less based on the raw materials. However, in the case of long-term intake for health purposes, the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

There is no particular limitation on the type of dietary supplement. Examples of foods to which the health functional food composition may be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products, including ice cream, various soups, drinks, tea Drinks, alcoholic beverages, vitamin complexes, and the like, and include all health foods in the conventional sense.

In addition, the nutraceutical composition of the present invention may be prepared as a food, in particular a functional food. The functional food of the present invention may include ingredients that are commonly added. Examples include proteins, carbohydrates, fats, nutrients and seasonings. For example, when prepared with a drink, natural carbohydrates or flavors may be included as additional ingredients in addition to the active ingredient. The natural carbohydrates can be monosaccharides (e.g. glucose, fructose, etc.), disaccharides (e.g. maltose, sucrose, etc.), oligosaccharides, polysaccharides (e.g. dextrins, cyclodextrins, etc.) or sugar alcohols (e.g. , Xylitol, sorbitol, erythritol and the like). The flavoring agent may be a natural flavoring agent (eg, taumartin, stevia extract, etc.) and synthetic flavoring agents (eg, saccharin, aspartame, etc.).

Various nutritional supplements, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols, carbonic acid The carbonation agent etc. which are used for a drink can be contained further. Although the ratio of the above-mentioned ingredients is not critical, it is generally selected from 0.01 to 0.1 parts by weight based on 100 parts by weight of the health functional food composition of the present invention.

In addition, the present invention relates to a pharmaceutical composition for the prevention or treatment of obesity containing the shellfish extract as an active ingredient.

The pharmaceutical compositions of the present invention may further comprise suitable carriers, excipients or diluents commonly used in the manufacture of pharmaceutical compositions.

Pharmaceutical dosage forms of the compositions according to the invention can be used alone or in combination with other pharmaceutically active compounds, as well as in any suitable collection.

The pharmaceutical compositions according to the invention can be used in the form of oral dosage forms, external preparations, suppositories, and sterile injectable solutions, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., respectively, according to conventional methods. Can be. Carriers, excipients and diluents that may be included in the pharmaceutical composition comprising the clam extract include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate Various compounds or mixtures, including calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. have. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations include at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. Mix is prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups. In addition to the commonly used simple diluents, water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, utopsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The preferred dosage of the pharmaceutical composition of the present invention depends on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art.

The shellfish extract of the present invention can be administered to various mammals such as mice, mice, livestock, humans, and the like. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

Hereinafter, the present invention will be described in more detail with reference to Preparation Examples and Examples. These preparations and examples are only intended to explain the present invention more specifically, it is apparent to those skilled in the art that the scope of the present invention is not limited thereto.

Production Example  1. Preparation of Shellfish Extract

Shellfish used as a raw material of the present invention ( Scapharca broughtonii ) purchased and used biological products at Beolkyo Komak Co., Ltd. That is, in December 2017, it was taken from Beolgyo-eup, Boseong-gun, Jeonnam, and frozen in 1kg units and stored at -40 ℃ until used in the experiment.

The shellfish raw material was thawed for about 4 hours at 1 kg unit at room temperature before extraction, and then washed three times quickly in 10-fold (w / v) tap water, followed by desalting. The NaCl content was self-tested using a salinity meter. . The shellfish raw material of which desalination was completed was used after being shelled off.

1) Preparation of Shellfish Ethanol Extract (Experimental Example 1)

The thawed and desalted shells were ground using a blender (7012S, Waring, USA). 100 g of the ground clam was immersed in 1 l of 95% (v / v) ethanol, and extracted for 3 hours at 25 ± 5 ° C. and 120 rpm on a shaking incubator (DA-SIM-T, Dong a, Korea). It was. Thereafter, the filtrate was filtered using a filter paper (Watman No. 2), the filtrate was removed from the organic solvent on a rotary evaporator (N-100v-W, Eyela, Japan), and the water layer was freeze-dried (FDS8508, I; shin). , biobase, Korea).

2) Preparation of shellfish hydrothermal extract (Comparative Example 1)

The thawed and desalted shells were ground using a blender (7012S, Waring, USA). 100 g of the ground clam was immersed in 1 L of distilled water, and then placed in a reflux extractor having a capacity of 2 L and extracted for 3 hours in a heating mantle (Tops, MS-E106) at 100 ° C. Thereafter, the filtrate was filtered using filter paper (Watman No. 2) and the filtrate was lyophilized in a freeze dryer (FDS8508, I; shin, biobase, Korea).

3) Preparation of Extract (Comparative Example 2) of Shellfish Protamex Hydrolyzate

After desalting, 100 g of the shelled shellfish was heated in 1 L of boiling water for 5 minutes. The heated clam was pulverized using a mixer (7012S, Warning, USA), and then the pulverized product was placed in a 2 L round media reagent bottle and immersed in 1 L distilled water. 1 g of protamex (1.5AU / g) was added to the immersed shellfish sample and reacted in a 40 ° C. constant temperature water bath (BF-10BF, biofree) for 3 hours, followed by an autoclave (AC-14, JEIO TECH). , Korea) for 30 minutes at 100 ℃ to inactivate the enzyme. After completion of the reaction, the hydrolyzate was filtered using filter paper (Watman No. 2), followed by adding 2 parts by volume of 60% (v / v) ethanol to 1 part by volume of the filtrate, followed by centrifugation (3000 × g, 10 minutes). ) To obtain a supernatant. Thereafter, the mixture was concentrated under reduced pressure to remove the organic solvent of the supernatant, and then lyophilized in a freeze dryer (FDS8508, I; shin, biobase, Korea).

4) Preparation of extract of Comparative Shell Nutrase Hydrolyzate (Comparative Example 3)

After desalting, 100 g of the shelled shellfish was heated in 1 L of boiling water for 5 minutes. The heated clam was pulverized using a mixer (7012S, Warning, USA), and then the pulverized product was placed in a 2 L round media reagent bottle and immersed in 1 L distilled water. 1 ml of Neutrase 0.81 was added to the immersed clamshell sample and reacted in a 50 ° C. constant temperature water bath (BF-10BF, biofree) for 3 hours. After completion of the enzyme reaction, the hydrolyzate was autoclaved (AC-14). , JEIO TECH, Korea) was used for 30 minutes at 100 ℃ to inactivate the enzyme. After completion of the reaction, the hydrolyzate was filtered using a filter paper (Watman No. 2), followed by adding 2 parts by volume of 60% (v / v) ethanol to 1 part by volume of the filtrate, followed by centrifugation (3000 × g, 10 Min) to obtain supernatant. Thereafter, the mixture was concentrated under reduced pressure to remove the organic solvent of the supernatant, and then lyophilized in a freeze dryer (FDS8508, I; shin, biobase, Korea).

5) Preparation of extract of Comparative Shell Protamex and Neutrase Hydrolysate (Comparative Example 4)

After desalting, 100 g of the shelled shellfish was heated in 1 L of boiling water for 5 minutes. The heated clam was pulverized using a mixer (7012S, Warning, USA), and then the pulverized product was placed in a 2 L round media reagent bottle and immersed in 1 L distilled water. 1 g of protamex (1.5AU / g) was added to the immersed shellfish sample and reacted in a 40 ° C. constant temperature water bath (BF-10BF, biofree) for 3 hours, followed by an autoclave (AC-14, JEIO TECH). , Korea) for 30 minutes at 100 ℃ to inactivate the enzyme. Thereafter, 1 ml of neutrase (neutrase 0.81) was added to the clam protamex hydrolysate in which protamex was inactivated and reacted in a 50 ° C. constant temperature water bath (BF-10BF, biofree) for 3 hours, followed by autoclave. (AC-14, JEIO TECH, Korea) using the reaction at 100 ℃ 30 minutes to inactivate the enzyme. After completion of the reaction, the clam protamex and Neutrase hydrolyzate were filtered using filter paper (Watman No. 2), followed by adding 2 parts by volume of 60% (v / v) ethanol to 1 part by volume of the filtrate, and then centrifuging The supernatant was obtained by separation (3000 x g, 10 minutes). Thereafter, the mixture was concentrated under reduced pressure to remove the organic solvent of the supernatant, and then lyophilized in a freeze dryer (FDS8508, I; shin, biobase, Korea).

Example 1. Confirmation of cytotoxicity of shellfish extract

Toxicity of the 3T3-L1 cell line, which is an adipose progenitor cell, of each clam extract of Preparation Example 1 was measured by MTT colorimetric assay using 3T3-L1 (Korea Cell Line Bank, 10092.1, Korea), a progenitor cell. It was.

The 3T3-L1 cell line was dispensed in 200 μl at a concentration of 4 × 10 ⁴ cells / ml in a 96 well plate and incubated at 37 ° C. and 5% CO ₂ for 24 hours. Thereafter, after removing the culture medium, 180 μl of DMEM (Dulbeco's Modified Eagle's Media) containing 10% (v / v) FBS was added, and each of the shell extracts of Preparation Example 1 had a final concentration of 100, 500, or 1000 μg / 20 μl each was added to make the mL. After treatment, the cells were cultured for 24 hours and then treated with MTT reagent prepared at a concentration of 2 μg / ml at 2 mg / ml, and reacted for 3 hours, and then 540 nm absorbance was measured on a microplate reader to confirm cell viability.

As a result, as shown in Figure 1, each of the extracts of Preparation Example 1 was a safe substance that does not exhibit toxicity even at a concentration of 1000 µg / ml.

Example  2. Inhibitory Effect of Shellfish Extract on Adipocyte Differentiation

Determination of adipocyte differentiation inhibitory activity of each shell extract of Preparation Example 1 was cultured in 12-well plate of fat precursor cells 3T3-L1, until the cells are filled with a single layer on the bottom of the culture vessel (confluent) The medium was then changed and 2 more days incubated (post-confluent state). Then, each concentration of shellfish extract of 0.5 mM IBMX, 1 μM dexamethasone, 10 μg / ml insulin, and Preparation Example 1 was added to DMEM to which 10% (v / v) FBS (fetal bovine serume) was added. Treatment induced differentiation. Two days after the induction of differentiation, the culture solution containing only 10 μg / ml insulin and each of the shell extracts of Preparation Example 1 were treated by concentration, and every two days thereafter, in the culture medium containing no insulin until the sixth day after induction of differentiation. Cells were cultured by treating each shell extract of Example 1 by concentration. On day 8 from the start of differentiation, the medium was removed and the cell culture was washed with PBS (phosphate-bufferd saline (1X)), and then 4% (v / v) formaldehyde (ml) was added to each well to fix the cells. I was. After cell fixation, formaldehyde was removed, washed with PBS, and 1 ml of Oil Red O working solution was added to each well and stained at room temperature for 30 minutes. After staining three times with PBS, washed with 100 μl of 100% DMSO (dimethyl sulfoxide) was added to elute the lipids (lipid) accumulated in the cells, and transferred to a 96 well plate was measured for absorbance at 510nm. Absorbance values are expressed as percentages compared to the Control (untreated) control.

As a result, as shown in FIG. 2 and Table 1, the clam ethanol extract (Experimental Example 1) showed the effect of inhibiting adipocyte differentiation depending on the treatment concentration, but the clam water extract (Comparative Example 1), the extract of clam hydrolyzate ( In Comparative Example 2 to Comparative Example 4) there was no inhibitory effect on adipocyte differentiation.

Adipocyte Differentiation Rate (% of Control) IC ₅₀ value (μg / ml) 500 µg / ml 1000 µg / ml Experimental Example 1 71.19 ± 0.45 48.45 ± 0.57 924 ± 9.61 Comparative Example 1 115.88 ± 2.04 97.26 ± 4.13 > 1000 Comparative Example 2 125.97 ± 7.81 103.74 ± 14.21 > 1000 Comparative Example 3 109.15 ± 9.4 108.15 ± 0.06 > 1000 Comparative Example 4 114.91 ± 2.83 100.06 ± 2.77 > 1000

Claims (6)

Anti-obesity health functional food composition containing Scapharca broughtonii ethanol extract as an active ingredient. delete According to claim 1, wherein the composition is an anti-obesity health functional food composition, characterized in that to inhibit the differentiation of fat cells. According to claim 1, wherein the composition is an anti-obesity dietary supplement composition, characterized in that the formulation is made of any one selected from powder, granules, pills, tablets, capsules, candy, syrups and beverages. Pharmaceutical composition for the prevention or treatment of obesity containing the shellfish ethanol extract as an active ingredient. The pharmaceutical composition for preventing or treating obesity according to claim 5, further comprising a pharmaceutically acceptable carrier, excipient or diluent in addition to the shellfish extract.

Images