KR20230014156A - Composition for Anti-Obesity Comprising Catechin, and Complex Extracts of Rosa davurica Pall as Active Ingredient - Google Patents

Abstract

The present invention relates to an anti-obesity composition containing an extract of Rosa davurica as an active ingredient. The anti-obesity composition of the present invention exhibits excellent antioxidant effects, effectively inhibits α-amylase enzyme and pancreatic lipolytic enzyme, inhibits differentiation of preadipocytes, and reduces cholesterol and neutral fat in the blood. Accordingly, the present invention has excellent effects in the treatment and prevention of obesity. In addition, the anti-obesity composition of the present invention is not only very safe for the human body as the composition is made from an extract of Rosa davurica, which is a natural plant material, but also has excellent stability. Thus, the composition can be used in the food and pharmaceutical fields related for the treatment and prevention of obesity.

Description

Composition for Anti-Obesity Comprising Catechin, and Complex Extracts of Rosa davurica Pall as Active Ingredient}

The present invention relates to an anti-obesity composition comprising an extract of wild genus japonica as an active ingredient.

Obesity is a condition in which body fat is excessively accumulated to the extent that it has a detrimental effect on health. Obesity is defined as when body fat is more than 25% of body weight in men and 30% or more in women.

Obesity is caused by diseases such as dysfunction of the hypothalamus and abnormal energy metabolism, as well as genetic factors, but most obesity is caused by lifestyles caused by excessive nutrient intake and reduced physical activity. In recent years, the consumption of instant food has increased due to the convenience of life and western eating patterns, and the lack of exercise has increased, resulting in a continuous increase in the incidence of obesity, and it is expected that this trend will intensify over time.

The biggest problem of obesity is not simple obesity itself, but various metabolic diseases such as diabetes, hyperlipidemia, heart disease, stroke, arteriosclerosis, fatty liver, etc. that occur as obesity continues for a long time. In addition, obesity increases the risk of various diseases such as infertility, menstrual irregularity, degenerative arthritis, some cancers, sleep apnea, respiratory disorders, cholelithiasis, and depression, which is a risk factor and social problem of adult diseases.

Recently, by socially recognizing obesity as a disease, various pharmaceutical companies are accelerating the development of drugs to treat obesity. Xenical, a fat absorption inhibitor, is one of the most used obesity treatments worldwide. Orlistat, a component of Xenical, binds to digested fat and inhibits absorption in the intestines, thereby excreting part of the fat component as it is during meals. Other drugs that have been developed include reductyl and exolyze, which promote satiety.

However, when the commercialized drugs are taken for a long time, side effects including liver damage, gastrointestinal bleeding, pancreatitis, and kidney stones have been reported, and it is known that there is a risk of developing heart disease, respiratory disease, and nervous system disease.

Therefore, as natural products with fewer side effects and improved therapeutic effects have recently received attention, natural extracts for the prevention or treatment of obesity have been developed, and Codium jade plants and Astragalus extracts (Patent Registration KR No. 10-2250815), Aral Leah excelsa extract and its fractions (patent registration KR 10-2246783), Astragalus extract and Borage extract (patent registration KR 10-2242328), dogwood leaf extract (patent registration KR 10-2232867) Although the anti-obesity effect of the plant material is known, there is no known treatment or prevention effect of obesity disease on the raw fever tree.

Under this background, the inventors of the present inventors have made diligent research efforts to clarify the functionality in the process of conducting research on the usefulness of raw wild chimney extract. , It has an excellent effect in inhibiting the differentiation of preadipocytes, and has an effect of reducing total cholesterol and triglyceride content, so it was confirmed that there is a distinct activity in anti-obesity activity, and the present invention was completed.

Accordingly, the main object of the present invention is to provide a composition for anti-obesity containing the extract of wild genus japonica as an active ingredient.

In addition, another object of the present invention is to provide a food composition containing as an active ingredient an extract of wild chives exhibiting an anti-obesity effect.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a composition for anti-obesity comprising the extract of the green fever tree as an active ingredient.

The present inventors conducted a study on the usefulness of the components of the extracts of the wild cypress tree, and as a result, the cypress tree extract had excellent antioxidant effects, significantly inhibited the activity of α-amylase enzyme and pancreatic lipolysis enzyme, and preadipocyte cells (preadipocyte). ), and it was confirmed that there is an excellent effect of reducing the total cholesterol and triglyceride content, and the present invention was completed.

The composition of the present invention is a composition for the prevention and treatment of obesity disease, which contains as an active ingredient an extract of a natural plant derived from a safe natural plant material without side effects, and is safe without side effects even when applied to the human body.

" Rosa davurica Pall." of the present invention means a deciduous shrub of the dicotyledonous plant Rosaceae Rosaceae. In a preferred embodiment of the present invention, it is found that the extract of the wild chives contains a large amount of Quercetin, Kaempferol 3,4-di-O-glucoside, Ellagic acid, and Apigenin. have confirmed

As used in the present invention, the term "extract" refers to an extract obtained by the extraction treatment of a raw heathen tree, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, a refined or purified product of the extract, or a mixture thereof, etc. , including the extract itself and extracts of all formulations that can be formed using the extract. The extract of the present invention may be preferably prepared and used in the form of a dry powder after extraction. In the extraction of the fresh wild cypress tree, the method for extracting the extract is not particularly limited, and can be extracted according to a method commonly used in the art. Non-limiting examples of the extraction method include a hot water extraction method, an ultrasonic extraction method, a filtration method, a reflux extraction method, and the like, which may be performed alone or in combination with two or more extraction methods.

In the present invention, the type of extraction solvent used for extracting the natural heat guava tree is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the extraction solvent include water; C1 to C4 lower alcohols such as methanol, ethanol, propyl alcohol and butyl alcohol; polyhydric alcohols such as glycerin, butylene glycol, and propylene glycol; and hydrocarbon solvents such as methyl acetate, ethyl acetate, acetone, benzene, hexane, diethyl ether, and dichloromethane; Or a mixture thereof may be used, and preferably, water, lower alcohol, 1,3-butylene glycol, and ethyl acetate may be used alone or in combination of two or more.

In the present invention, the extract obtained by hot water extraction or cold brewing is filtered to remove floating solid particles by filtering out the particles using, for example, nylon or by using cryofiltration, and then used as it is or freeze-dried, hot-air-dried, It can be used after being dried using spray drying or the like.

As used herein, the term "fraction" refers to a product obtained by performing fractionation to separate a specific component or a specific component group from a mixture containing various components.

As a non-limiting example of the fractionation method, a method of obtaining a fraction from the extract by treating the extract obtained by extracting the extract from the extract with a predetermined solvent may be mentioned. In the present invention, the type of solvent used to obtain the fraction is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the fractionation solvent include polar solvents such as water and alcohol; and non-polar solvents such as hexane, ethyl acetate, chloroform, and dichloromethane. These may be used alone or in combination of two or more. When alcohol is used among the fractionation solvents, C1 to C4 alcohols may be specifically used.

The extract of the wild cypress tree of the present invention can be extracted from any one or more parts of the whole plant, leaf, stem, root, and fruit of the cypress tree. In addition, the extract of the wild plant extract can be extracted from natural, hybrid or mutant plants, and can also be extracted from plant tissue culture.

In the present invention, the raw yeolgi tree extract is characterized in that it exhibits a preventive or therapeutic activity of obesity disease by itself.

The term "prevention" of the present invention refers to any action that inhibits or delays the onset of obesity or obesity-related diseases by administration of the composition according to the present invention, and the term "treatment" refers to the obesity by administration of the composition according to the present invention. or any action that improves or beneficially alters the symptoms of an obesity-related disease.

In a preferred embodiment of the present invention, the extract of the wild chives has an excellent antioxidant effect, increases the α-amylase enzyme and pancreatic lipolytic enzyme inhibitory activity, increases the preadipocyte differentiation inhibitory effect, and increases the total It has been confirmed that cholesterol and triglyceride content are reduced, thereby having an excellent effect on anti-obesity.

The composition for anti-obesity containing the extract of the present invention as an active ingredient is characterized by having antioxidant activity. In a preferred embodiment of the present invention, it was observed that the experimental group treated with the extract of the present invention exhibited an overall superior antioxidant effect compared to the negative control group. These antioxidant effect values were analyzed to be at a similar level when compared to the positive control group, the ascorbic acid-treated group (FIGS. 3 and 4). In addition, it was confirmed that the extract of wild lentil tree extract exhibited an excellent cytoprotective effect against oxidative stress even in adipocytes (FIG. 5).

In addition, the anti-obesity composition of the present invention is characterized by inhibiting the activity of α-amylase enzyme and pancreatic lipolytic enzyme. In a preferred embodiment of the present invention, it was confirmed that the α-amylase enzyme inhibitory activity increased by 66% or more at the concentration of 250 μg/ml of the extract of the present invention (Fig. 6), compared to the negative control group, in all sections of the pancreas. It was confirmed that the inhibitory activity of the lipase enzyme was increased, and in particular, it was confirmed that the extract of the present invention was increased by 47.1% at a concentration of 250 μg / ml (FIG. 7).

The anti-obesity composition of the present invention is characterized by having anti-obesity activity through inhibition of differentiation of preadipocytes. In a preferred embodiment of the present invention, a 36.5% improvement in the adipocyte differentiation inhibitory effect was confirmed in the group treated with 250 μg/ml of the fresh wild chive extract of the present invention (FIG. 8).

In addition, the anti-obesity composition of the present invention is characterized in that it has anti-obesity activity through the effect of reducing serum total cholesterol. In a preferred embodiment of the present invention, it was confirmed that the total cholesterol reduction effect was 8.18% and 15.5%, respectively, in the groups treated with 100 and 200mg/kg of the extract of the present invention (Fig. 9).

The anti-obesity composition of the present invention is characterized by having anti-obesity activity through serum triglyceride concentration. In a preferred embodiment of the present invention, the neutral fat reduction effect of 42.5% and 47.5%, respectively, was confirmed in the treatment groups of 100 and 200mg/kg of the fresh wild chive tree extract of the present invention (FIG. 10).

According to one aspect of the present invention, the present invention provides a pharmaceutical composition for anti-obesity comprising an extract of japonica japonica as an active ingredient. The pharmaceutical composition of the present invention has excellent antioxidant effect, excellent inhibitory activity of α-amylase enzyme and pancreatic lipolytic enzyme, excellent effect on preadipocyte differentiation inhibitory effect and reduction of total cholesterol and triglyceride content Since there is, it can be usefully used as an anti-obesity pharmaceutical composition for the purpose related to the treatment and prevention of obesity.

On the other hand, the pharmaceutical composition for anti-obesity containing the extract of the present invention as an active ingredient may be prepared in the form of a pharmaceutically acceptable salt.

Specifically, a salt may be formed by adding an acid, for example, an inorganic acid (eg, hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid, etc.), an organic carboxylic acid (eg, acetic acid, halo such as trifluoroacetic acid) Acetic acid, propionic acid, maleic acid, succinic acid, malic acid, citric acid, tartaric acid, salicylic acid), and acidic sugars (glucuronic acid, galacturonic acid, gluconic acid, ascorbic acid), acidic polysaccharides (e.g. hyaluronic acid, chondroitin sulfate) , arginic acid), organic sulfonic acids including sulfonic acid sugar esters such as chondroitin sulfate (eg methane, sulfonic acid, p-toluene sulfonic acid), etc. may be added to form salts.

The pharmaceutical composition of the present invention consists of materials separated from natural extracts that are not cytotoxic, and can be administered orally or parenterally during clinical administration, and can be used in the form of general pharmaceutical preparations.

That is, the pharmaceutical composition of the present invention can actually be administered in various oral or parenteral formulations. When formulated, commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants are used. It is prepared by Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, liurine fat, glycerogeratin, and the like may be used.

In addition, the pharmaceutical composition of the present invention may be mixed with various pharmaceutically acceptable carriers such as physiological saline or organic solvents, and may be mixed with carbohydrates such as glucose, sucrose or dextran to increase stability or absorption. , antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers can be used as pharmaceuticals.

The effective dose of the pharmaceutical composition of the present invention is 0.01 mg/kg to 10 mg/kg, preferably 0.1 mg/kg to 1 mg/kg, and may be administered once to three times a day.

The total effective amount of the pharmaceutical composition of the present invention can be administered to the patient in a single dose in the form of a bolus or by infusion for a relatively short period of time, and can be administered in multiple doses. This may be administered by a fractionated treatment protocol in which administration is administered over a long period of time. Since the effective dose of the patient is determined considering various factors such as the patient's age and health condition as well as the drug administration route and number of treatments, considering this point, those of ordinary skill in the art can use the present invention It will be possible to determine an appropriate effective dosage according to the particular use as a pharmaceutical composition of the.

According to another aspect of the present invention, the present invention provides a food composition for anti-obesity comprising the extract of the plant as an active ingredient. The extract of the present invention is as described above. The food composition may be used in the form of health functional food, but is not limited thereto.

The food composition of the present invention may be included in the form of an extract of Saengyeolgi tree, a fraction thereof, or a processed product thereof. In addition, the composition may include a food additive that is acceptable in food science in addition to the active ingredient.

In the present invention, "food supplement additive" refers to a component that can be added to food supplementally, and can be appropriately selected and used by those skilled in the art as being added to prepare a health functional food of each formulation. Examples of food additives include various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and fillers, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners , pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages, etc. are included, but the types of food additives of the present invention are not limited by the above examples.

The food composition of the present invention may include health functional food. In the present invention, "health functional food" refers to food manufactured and processed in the form of tablets, capsules, powders, granules, liquids and pills using raw materials or ingredients having useful functionality for the human body. Here, "functionality" means obtaining useful effects for health purposes, such as adjusting nutrients for the structure and function of the human body or physiological functions. The health functional food of the present invention can be prepared by a method commonly used in the art, and can be prepared by adding raw materials and components commonly added in the art during the preparation.

In addition, the formulation of the health functional food may also be manufactured without limitation as long as the formulation is recognized as a health functional food. The food composition of the present invention can be prepared in various types of formulations, and unlike general drugs, it has the advantage of using food as a raw material and has no side effects that may occur when taking drugs for a long time, and has excellent portability, improving obesity It can be taken as an adjuvant to enhance its effect.

There is no limit to the form that the health functional food of the present invention can take, and it can include all foods in a conventional sense, and it can be used interchangeably with terms known in the art, such as functional foods. In addition, the health functional food of the present invention may be prepared by mixing suitable other auxiliary ingredients and known additives that may be included in food according to the selection of those skilled in the art. Examples of foods that can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and Vitamin complexes, etc., can be prepared by adding the compound represented by Formula 1 according to the present invention to juice, tea, jelly, juice, etc. prepared as a main component. Also included are foods used as feed for animals.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a composition for anti-obesity comprising the extract of wild genus japonica as an active ingredient.

(b) The anti-obesity composition of the present invention exhibits excellent antioxidant effect, has excellent inhibitory activity of α-amylase enzyme and pancreatic lipolytic enzyme, inhibits preadipocyte differentiation, and has high total cholesterol and triglyceride content. It has an excellent effect on reduction.

(c) In addition, the anti-obesity composition of the present invention is not only very safe to the human body, but also has excellent stability, as it is made from natural plant extracts, the natural plant extract, and is very safe for the human body. It can be usefully used for related anti-obesity purposes.

Figure 1 is a graph of the chromatogram results of the extracts of wild cypress (rosehip) for each part (A: standard mix, B: radishes, C: leaves, D: radishes, stems, E: radishes). Root, F: fresh sage fruit, peak 1: Ellagic acid, peak 2: Kaempferol 3,4-di-O-glucoside, peak 3: Quercetin).
Figure 2 is a graph showing the measurement of cell viability of 3T3-L1 cells, which are pre-adipocytes, according to the concentration of the extract of the present invention.
Figure 3 is a graph showing the measurement of DPPH radical scavenging activity according to the treatment concentration of the extract of the present invention.
Figure 4 is a graph showing the measurement of ABTS radical scavenging activity according to the treatment concentration of the extract of the present invention.
5 is a graph showing intracellular ROS scavenging activity in 3T3-L1 cells, which are pre-adipocytes, according to the treatment concentration of the extract of the present invention.
Figure 6 is a graph showing the α-amylase enzyme activity inhibition rate measured according to the treatment concentration of the extract of the present invention.
Figure 7 is a graph showing the measurement of the pancreatic lipolytic enzyme activity inhibition rate according to the treatment concentration of the raw heat chimney extract of the present invention.
8 is a graph showing the inhibition of preadipocyte differentiation in 3T3-L1 cells, which are pre-adipocytes, according to the treatment concentration of the fresh tropical fruit extract of the present invention.
Figure 9 is a graph showing the measurement of the total cholesterol content according to the concentration of the treatment of the extract of the present invention in an animal model induced with a high-fat diet.
Figure 10 is a graph showing the measurement of the neutral fat content according to the treatment concentration of the raw pyrrhiza sinensis extract of the present invention in an animal model induced with a high-fat diet.

Hereinafter, the present invention will be described in more detail through examples. Since these examples are intended to illustrate the present invention only, the scope of the present invention is not to be construed as being limited by these examples.

Example 1. Preparation of extracts of fresh lentil trees

Preparation Example 1-1. Preparation of extracts from wild plants

After removing impurities by washing the outposts of the wild plants (origin: domestic, place of purchase: Wonsam Rosehip Co., Ltd.) in running water, 1L of 30% ethanol aqueous solution was added to 100 g of the plants, stirred at room temperature for 1 day, and then stirred for 1 hour. Extracted by sonication. The process was carried out in 3-batch. After collecting the obtained extract, concentrated under reduced pressure at 40 ° C. or less, and lyophilized to prepare a powder form (Preparation Example 1-1).

Preparation Example 1-2. Manufacture of extracts of wild genus japonica leaves

Except for the use of the leaves of the wild cypress tree, the extract of the leaf extract of the cypress tree was prepared in the same manner as in Preparation Example 1-1.

Preparation Example 1-3. Manufacture of the extract of the stem of the wild apricot tree

Except for using the stem of Cyperus japonica, the extract of the stem of Cyperus japonica was prepared in the same manner as in Preparation Example 1-1.

Preparation Example 1-4. Preparation of the extract of the root of the wild apricot tree

Except for the use of the root of Cyperus japonica, the extract of the root of Cyperus japonica was prepared in the same manner as in Preparation Example 1-1.

Preparation Example 1-5. Preparation of extracts from the fruit of the wild apricot tree

A fruit extract of the fruit of the fruit of the wild plant was prepared in the same manner as in Preparation Example 1-1, except that the fruit of the plant of the wild plant was used.

Example 2. Component analysis of the extract of fresh genus japonica

The content of the indicator component of the extracts of the wild genus japonica prepared in Example 1 was compared and analyzed.

High performance liquid chromatography analysis was performed using Thermo UHPLC U3000. HPLC analysis conditions were as follows. The column used was a Sunfire TM C18 column (Waters, 250 × 4.6 mm, 5 μm), and the column temperature was 30 ° C. The UV wavelength was measured at 280 nm, and water containing 0.1% formic acid and acetonitrile (gradient mode) were used as the mobile phase. [0040] The fresh rhododendron extract (Preparation Examples 1-1 to 1-5) prepared in Example 1 was dissolved in a mobile phase solvent at a concentration of 1 mg/ml, and 10 uL was injected, followed by measurement at a flow rate of 1 ml/min. The content of the marker component of the extract is shown in [Table 1].

[Table 1]

Referring to [Table 1] and [Fig. 1], the outposts of the genus japonica are rich in ellagic acid, kaempferol 3,4-di-O-glucoside, and quercetin. It was analyzed as containing some Apigenin.

In addition, unlike other parts of the plant, the leaves of the green fever plant contain ellagic acid, quercetin, and apigenin, and the roots of the green fever plant contain ellagic acid and quercetin. analyzed.

Experimental Example 1. Cytotoxicity of the extracts of fresh fever

The cytotoxicity of the extract of the present invention prepared in Example 1 against 3T3-L1 cells was confirmed. Specifically, 3T3-L1 preadipocytes (ATCC CL-173, American Type Culure Collection, Manassas, USA) were cultured using DMEM containing 10% bovine calf serum, and differentiation induction and DMEM containing 10% fetal bovine serum was used as a maturation medium, and cultured in an incubator at 37°C and 5% CO ₂ conditions. After culturing in a 96-well plate for cell culture to confluent state, the extract prepared in Preparation Example 1-1 was treated at concentrations of 1, 10, 50, 100, and 250 μg/ml, respectively, After 48 hours, MTT (tetrazolium bromide salt) was treated and incubated for 2 hours. After culturing, all the culture medium was removed, and formazin produced by dispensing 100 μl of DMSO was dissolved, and absorbance was measured at 570 nm (determination wave) and 690 nm (reference wave) wavelengths using a microplate reader.

Referring to Figure 2, it was confirmed that the cell survival rate of 95% or more was confirmed in all concentrations of the extract of the present invention, and it was confirmed that it did not exhibit cytotoxicity.

Experimental Example 2. Antioxidant activity of the extracts of fresh wild genus japonica

2-1. DPPH radical scavenging activity

Oxidative stress activates the signal transduction system of nerve cells, increases the generation of reactive oxygen species such as hydrogen peroxide, and reduces the expression of antioxidant enzymes. The increase in reactive oxygen species changes the structure of genes and proteins, destroys intracellular and extracellular homeostasis, and causes damage to nerve cells. The inhibitory activity of DPPH radical, one of the reactive oxygen species that causes damage to nerve cells, is an important index for evaluating antioxidant efficacy.

Sang S et al., J. Agric. Food Chem. 50(8), p2459-2463, 2002) using the reducing power of DPPH (1,1-diphenyl-2-picrylhydrazyl, Sigma, USA) According to the method, the antioxidant activity of the extract of the present invention was measured.

After putting the extracts of wild silage plant prepared in Preparation Example 1-1 at concentrations of 1, 10, 50, 100, and 250 μg/ml, respectively, into a 96-well microplate and incubating at 37° C. for 30 minutes, the amount of DPPH reduced The absorbance was measured at 520 nm using a spectrophotometer (Precision microplate reader; Molecular Devices, VersaMax), and the activity was compared with that of a non-added control group to which no sample was added. The test was repeated three times, and the measurement results are shown in FIG. 3 . For comparative experiments, ascorbic acid was used as a positive control.

As a result of the experiment, it was observed that the experimental group treated with the extract of the present invention exhibited a very superior antioxidant effect compared to the negative control group, and these antioxidant effect values were equivalent to those of the positive control group treated with ascorbic acid. It was found to be above the level, and the antioxidant activity was analyzed to be very good.

2-2. ABTS radical scavenging activity

2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cation scavenging ability shows a unique blue-green color when ABTS radical is generated by the reaction of diammonium salt with potassium persulfide. It is a method to measure decolorization to light green as a sample showing antioxidant effect is added.

Antioxidant capacity measurement test using ABTS radical was performed by mixing 7 mM 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) and 2.4 mM potassium persulfide, blocking light for 16 hours before use, and incubating at room temperature. reacted After the reaction, 1, 10, 50, 100, and 250 μg / ml (in PBS, pH 7.4) was added and reacted, centrifuged after 10 minutes, and only the supernatant was obtained and absorbance was measured at 734 nm. For comparative experiments, ascorbic acid was used as a positive control.

As a result of the measurement, it was observed that the experimental group treated with the extract of the present invention exhibited a very superior antioxidant effect in all sections compared to the negative control group, and these antioxidant effect values were also observed when compared to the positive control group, ascorbic acid treated group. It was found to be at a similar level and was analyzed to exhibit very good antioxidant activity (FIG. 4).

In particular, it is noteworthy that it shows an antioxidant effect equivalent to that of the ascorbic acid-treated group, which is a positive control group, at a concentration of 50 μg/ml or higher. It is believed to be useful when

2-3. Intracellular ROS activity

In order to measure the amount of ROS production of adipocytes according to the differentiation process, 3T3-L1 preadipocytes (ATCC CL-173, American Type Culure Collection, Manassas, USA) were mixed with 5% FBS (fetal bovine serum), 10% horse It was inoculated into DMEM medium containing serum, 50 units/ml penicillin and 100 μg/ml streptomycin, and subcultured for 24-48 hours at 37°C, 5% CO ₂ conditions, and used for experiments.

3T3-L1 cells were dispensed at a concentration of 1.2×10 ⁵ to 10 ⁶ , and treated with the extract of the present invention (Preparation Example 1-1) at concentrations of 1, 10, 50, 100, and 250 μg/ml for 48 hours cultured for a while. After culturing, wash twice with sterilized PBS (pH7.4), add 80 μl of 0.2% NBT solution, react in a CO ₂ incubator for 90 minutes, and use a 7:3 mixture of DMSO and 1N KOH. Dark blue formazen was all eluted, and absorbance was measured at 570 nm using a micro plate. For comparative experiments, commercially available orlistat (tetrahydrolipstatin component) as an anti-obesity drug was used as a positive control group.

Referring to [Figure 5], which summarizes the experimental results, it was found that the extract of the present invention eliminates ROS production in 3T3-L1 cells in a concentration-dependent manner. In particular, it was found that radical generation in 3T3-L1 cells was reduced by 32.6% at a concentration of 250 μg/ml, and it was confirmed that the effect was similar to that of the positive control group. Through these experimental results, it was confirmed that the extract of the present invention exhibits an excellent cytoprotective effect against oxidative stress even in adipocytes.

Experimental Example 3. α-Amylase Inhibitory Activity of Fresh Yeolgi Tree Extract

In order to confirm the anti-obesity effect by controlling the blood glucose concentration after eating according to the inhibition of carbohydrate hydrolysis and absorption, the fresh genus japonica extract prepared in Example 1 (Preparation Example 1-1) is a glycolytic enzyme of α-amylase. The effect on activity was tested. Specifically, the extract of the present invention was mixed with 0.1 M sodium phosphate buffer (pH 6.9) and 0.5 unit/ml of α-amylase enzyme at concentrations of 1, 10, 50, 100, and 250 μg/ml, respectively. The solutions were mixed and incubated at 37°C for 10 minutes. After incubation, 200 μl of 5 mM [4-nitrophenyl-α-D-gluco-pyranoside] compound was added, reacted at 37 ° C for 5 minutes, and absorbance was measured at 405 nm using a microplate reader (Molecular Devices E09090, USA). measured. Orlistat (tetrahydrolipstatin component), which is commercially available as an anti-obesity drug, was used as a positive control.

Referring to [Figure 6], which summarizes the experimental results, it was confirmed that the extract of the present invention inhibited the activity of α-amylase by 87% or more at a concentration of 250 μg/ml, and had excellent α-amylase inhibitory activity. . Through the above experimental results, it was confirmed that the extract of the present invention can be used as a blood sugar control material as well as anti-obesity.

Experimental Example 4. Inhibition of Pancreatic Lipolytic Enzyme Activity of Fresh Ficus Tree Extract

Since fat is decomposed and absorbed by pancreatic lipase, inhibition of the activity of lipase enzyme can inhibit fat absorption in the blood.

Measurement of pancreatic lipase inhibitory activity was carried out by modifying the method performed by Kim et al. Specifically, porcine pancreatic lipase was dissolved in an enzyme buffer solution (10 mM MOPS, 1 mM EDTA, pH 6.8) at a concentration of 0.5 g/200 ml while maintaining 4° C., and the lysate was centrifuged at 4,000 rpm After that, the supernatant was mixed with Tris buffer (100 mM Tris-HCl, 5 mM CaCl ₂ , pH 7.0) and enzyme buffer. After dissolving the extract prepared in Preparation Example 1-1 in DMSO at concentrations of 1, 10, 50, 100, and 250 μg/ml, respectively, the mixture was stirred with the porcine pancreatic lipase enzyme at 37° C. for 15 minutes.

Thereafter, p-NPB (p-nitrophenyl butyrate) dissolved in DMF at a concentration of 10 mM was added and re-reacted at 37° C. for 35 minutes. After shaking the reaction, the absorbance was measured at 405 nm using an ELISA reader. Orlistat (tetrahydrolipstatin component), which is commercially available as an anti-obesity drug, was used as a positive control. The pancreatic lipase inhibitory activity was expressed as the absorbance activity inhibition rate of the sample solution added group and non-added group.

Referring to [Figure 7], which summarizes the experimental results, it was confirmed that the extract of the present invention has an excellent activity to inhibit pancreatic lipase enzyme in a concentration-dependent manner. It was confirmed that there is a very excellent effect of inhibiting more than %.

However, compared to the positive control group treated with orlistat, the inhibitory activity of the pancreatic lipase enzyme of the extract of the present invention was slightly lower than that of the positive control group. It has been reported that there are various side effects such as absorption inhibition. Therefore, it is expected that the extract of the present invention composed of natural ingredients can be effectively used as an alternative to anti-obesity drugs made of conventional compounds in that it inhibits the activity of pancreatic lipase enzyme without side effects.

Experimental Example 5. Adipocyte Differentiation Inhibitory Activity of Fresh Yeolgi Tree Extract

In order to confirm the anti-obesity effect of the extract of the present invention, 3T3-L1 preadipocytes (ATCC CL-173, American Type Culure Collection, Manassas, USA) were differentiated to determine the degree of fat accumulation in adipocytes. It was measured through Oil-red-O staining.

3T3-L1 cells were dispensed at a number of 5×10 ⁴ cells in a 24-well plate, and then maintained for 2 more days after the wells were 100% filled with cells. Adipocyte differentiation was induced for 2 days in 10% FBS DMEM medium containing MDI [0.5mM 3-isobutyl-1-methylxanthine (IBMX), 1μM dexamethasone, 1μM insulin], and after 48 hours of culture, 2μM insulin was added. It was cultured for two days in a medium containing 10% FBS DMEM.

It was replaced with 10% FBS DMEM culture medium for 4 days at 2-day intervals. During the induction of adipocyte differentiation, each culture was treated with the extract of the present invention prepared in Preparation Example 1-1 at concentrations of 1, 10, 50, 100, and 250 μg/ml, and at 8 On the first day, Oil-red-O staining was performed to confirm the degree of fat accumulation.

Referring to [Figure 8], which summarizes the experimental results, it was found that the extract of the present invention inhibits the differentiation of adipocytes in a concentration-dependent manner, and in the 250 μg/ml treatment group, 36.5% of adipocytes were reduced compared to the negative control group. A differentiation inhibition rate was observed and an excellent adipocyte differentiation inhibitory effect was observed.

However, compared to the positive control group treated with orlistat, the activity of inhibiting adipocyte differentiation of the extract of the present invention was measured to be somewhat lower. It has been reported that there are various side effects such as suppression.

Therefore, the extract of the present invention composed of natural ingredients is expected to be effectively used as an alternative to anti-obesity drugs made of conventional compounds in that it effectively inhibits adipocyte differentiation without side effects.

Experimental Example 6. Measurement of serum total cholesterol and triglyceride content using an animal model induced by a high-fat diet

Concentrations of 100 and 200 mg/kg/day of the high-fat diet and the extracts of the wild cypress tree in C57BL/6 experimental animals in which obesity was induced by feeding a high-fat diet was mixed and fed, and the effect on lipid components in the body was analyzed.

6-1. Breeding conditions and dietary composition of laboratory animals

As experimental animals, C57BL/6 5-week-old male mice with an average weight of 20 ± 1 g were used in the experiment after a 1-week adaptation period. The experimental group was divided into 4 groups of 5 so that each group had a similar body weight by the egg mass method, and all groups except for the normal diet group were fed a high-fat diet containing 60% of total calories as fat for 6 weeks to induce obesity.

The experimental environment was maintained at a temperature of 22 ± 1 ° C and humidity of 50 ± 5%, a light-dark cycle was adjusted to 12 hours, and water and food were freely fed during the breeding period. In addition, the breeding management of all laboratory animals was in accordance with the Laboratory Animal Use and Breeding Management Regulations, and the entire process of the experiment was conducted with the approval of the Animal Experimentation Ethics Committee of Kyung Hee University. The classification and dietary composition of the experimental group are summarized in [Table 2].

[Table 2]

6-2. Measurement of serum total cholesterol and triglyceride content

The total lipid content of serum was measured according to the method of Frings et al., after adding phospho-vanillin reagent to serum and incubating at 37 ° C for 15 minutes, measuring absorbance at 540 nm using the sample-free group as a control group, and calculated by a standard calibration curve. . Total cholesterol content was measured using a kit reagent (AM 202-k, Asan, Korea) for measuring total cholesterol, and triglyceride content was measured using a kit reagent (AM 157S-k, Asan, Korea) for measuring triglyceride.

Referring to [Figure 9], which summarizes the cholesterol content measurement results, the total cholesterol content of the ND group and the HFD group was measured to be 59.6 mg / dL and 109.2 mg / dL, respectively, indicating that the HFD group increased by about 84% compared to the ND group. appear. On the other hand, in the "HFD+100" group and the "HFD+200" group administered with the extract of the present invention, a reduction effect was observed in the range of about 8 to 15% compared to the HFD group.

In addition, referring to [Figure 10], which summarizes the triglyceride content measurement results, the total cholesterol content of the ND group and the HFD group was measured to be 43.6 mg / dL and 74.6 mg / dL, respectively, and the HFD group was about 71 compared to the ND group. showed a % increase. On the other hand, in the "HFD+100" group and the "HFD+200" group administered with the extract of the present invention, an excellent triglyceride reduction effect was observed, which was reduced to a level similar to that of the normal group.

Having described specific parts of the present invention in detail above, it is clear that these specific techniques are merely preferred embodiments for those skilled in the art, and the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (7)

An anti-obesity composition comprising an extract of wild genus japonica as an active ingredient. According to claim 1,
Anti-obesity composition, characterized in that the composition has an antioxidant activity. According to claim 1,
The composition is an anti-obesity composition, characterized in that inhibiting the activity of α-amylase enzyme and pancreatic lipolytic enzyme. According to claim 1,
Anti-obesity composition, characterized in that the composition exhibits anti-obesity activity through inhibition of differentiation of preadipocytes. According to claim 1,
The composition is an anti-obesity composition, characterized in that for reducing the content of cholesterol and triglycerides in the blood. According to any one of claims 1 to 5,
The composition is an anti-obesity composition, characterized in that the pharmaceutical composition. According to any one of claims 1 to 5,
The composition is an anti-obesity composition, characterized in that the food composition.

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