KR102513138B1 - Composition for Anti-obesity Using an Extract of Spiraea prunifolia - Google Patents

Abstract

The present invention suppresses the differentiation of 3T3-L1, a mouse preadipocyte, into adipocytes in a concentration-dependent manner, clearly suppresses weight gain in an obese animal model, and not only significantly reduces the amount of fat in visceral fat and subcutaneous fat, but also reduces liver fat Disclosed is an anti-obesity composition using bridal spirea extract having an activity of reducing the accumulation of subcutaneous fat, epididymal fat, and subperitoneal fat and reducing blood neutral fat.

Description

Anti-obesity composition using an extract of Spiraea prunifolia {Composition for Anti-obesity Using an Extract of Spiraea prunifolia}

The present invention relates to an anti-obesity composition using Spiraea prunifolia extract.

In modern society, the number of obese people is increasing due to a convenient living environment due to rapid automation, excessive nutritional intake due to an increase in processed foods and eating out, and a decrease in physical activity. In the United States, which has the highest obesity rate among developed countries, about 60% of adults are known to be overweight. As a percentage, it is steadily increasing compared to 26.0% in 1998, and this rate is increasing by 5% every 10 years, which is recognized as a serious problem threatening public health (J. R. Soc. Med. 99:250-257 2006). The World Health Organization (WHO) classifies obesity as a disease rather than a single phenomenon or symptom, and has reported that the world's obese population will increase to 1.5 billion in 2015 and become a serious health problem (Ann Epidemiol. 2005. 15:87-97; J Life Sci. 2013. 23:69-78). Although the cause of obesity is not clearly identified, it is not caused by a single condition, but by various causes such as improper diet and lifestyle, genetic factors, and reduced physical activity (.Korean J. Food Science Nutrition 22(1 ) 110-12.2009; Korean J. Food Science Nutrition 23(5):363-367.1990).

Obesity is an increase in the number and size of fat cells due to excessive accumulation of body fat due to an imbalance between energy intake and consumption (Cell.104:531-543 2001), and body energy is transferred to fat cells in the form of triglycerides. When the stored energy source is exhausted, the stored fat is decomposed into free fatty acids and glycerol to be used as an energy source, but excessive intake of energy promotes the differentiation of fat cells and increases the amount of stored fat in the body, which is a direct cause of obesity. (Metabolism. 30: 425-427 1981; Biochem J. 1;435(3):723-32 2011). Obesity acts as a risk factor that increases the incidence of various diseases as well as changes in body shape due to accumulation of internal and abdominal fat (Korean J. Pediatr.48:126-137. 2005). Metabolism becomes a problem, and symptoms such as abnormal secretion of hormones and abnormal secretion of cytokines occur. An increase in triglyceride and LDL-cholesterol, and a decrease in HDL-cholesterol due to obesity, lead to abnormal fat metabolism in the body, decrease the insulin receptor present in the tissue, and also reduce insulin sensitivity, inhibiting the transport of glucose into cells. It can also cause high blood sugar and diabetes. Obesity is also known to be closely related to the occurrence of metabolic diseases such as hyperlipidemia, cardiovascular disease, cancer, respiratory disorders, stroke, and osteoarthritis (Med. Int. 22 385-388 1994; Ann. Intern. Med 103:1024-1029 1985).

Physiological regulation of adipocytes can be largely divided into induction of differentiation of adipocytes, biosynthesis of fat, and decomposition of lipids. In the differentiation stage of adipocytes, various transcription factors such as PPAR (peroxisome proliferator activated receptor), C/EBP (CCAAT/enhancerbinding proteins), and SREBP (sterol regulatory element binding protein) are induced to express step by step in adipocytes undergoing differentiation. , Through the interaction of each factor, the expression of various adipocyte-specific genes is regulated (Genes Dev. 14:1293-1307. 2000). In adipocytes that have completed differentiation, triglyceride biosynthesis occurs using the fatty acids and glucose introduced into the fat cells. (acyl-CoA synthetase), diacylglycerol acyltransferase (DGAT), and the like (Trends Endocrinol. Metab., 23, 56-64. 2011; Biochem. Biophys. Res. Commun., 420,805-810. 2012). The neutral fat synthesized by the action of these enzymes is used as an energy source. Lipolysis is regulated by catecholamines and insulin depending on nutritional status. Enzymes involved in lipid degradation include ATGL (adipose triglyceride lipase), HSL (hormone sensitive lipase), and MGL (monoacyl glycerol lipase). It is divided into three molecules (Trends Endocrinol. Metab., 23, 56-64. 2011; Biochem. Biophys. Res. Commun., 420,805-810. 2012). Fatty acids produced by hydrolysis are oxidized in mitochondria and consumed as energy, or esterified and synthesized as neutral fats to be stored in cells.

Adipose adipocytes, 3T3-L1, were first isolated from 3T3 cells by Green and Meuth (Cell. 3(2):127-33. 1974), and their biological characteristics and the property of differentiating into adipocytes under appropriate culture conditions were revealed. It is widely used to conduct research on the differentiation process of adipocytes and the decomposition of accumulated fat. 3T3-L1 cells, which are pre-adipocytes, differentiate into mature adipocytes by various hormones and PPAR, C/EBP, and SREBP, and accumulate intracellular triglycerides by increasing the expression of related genes and related enzyme activities ( J Nutr 130: 3116S-3121S, 2000; J Nutr 130: 3122S-3126S, 2000). For research on the development of functional materials, the method of searching for materials that suppress the differentiation process or promote lipolysis using 3T3-L1 cells as described above is widely used with the aim of inhibiting excessive accumulation of neutral fat in adipose tissue.

Existing anti-obesity drugs such as orlistat and sibutramine are known to have serious side effects such as vomiting, constipation, gastrointestinal disorders, and cardiovascular diseases (Int J Obes Relat Metab Disord. Jul; 25(7)). : 1095-9.2001; Obes Res. 8(6):431-7.2000; Lancet. 6;369(9555):71-7.2007; Front Physiol. Efforts to develop effective and safe materials are ongoing. Retinol, vitamin E, vitamin U, Japanese pepper extract, etc. have been reported as substances that inhibit the differentiation of fat cells (Mol Cell Biol. 16:15671575. 1996;Ann Dermatol. Feb;24(1):39- 44 2012: J Nutr. .

Meadowsweet ( Spiraea prunifolia for. simpliciflora Nakai) is a medicinal plant belonging to the rose order, and is widely distributed in domestic mountains nationwide and grows well in sunny places such as foothills and fields. In general, in traditional medicine, the root is used as a medicinal material, and it can be used for antipyretic, contraction of vascular tissue, and convergence of branches. It can also be used for symptoms such as fever caused by a cold, neuralgia, swelling and pain in the throat, ailments (symptoms of chills and fever rising at a certain time every day), and diarrhea.

Nevertheless, research on the pharmacological components and uses of meadowsweet has not been carried out much, so the application of meadowsweet is not wide.

Specifically, Korean Registered Patent No. 10-1176526 discloses the anti-aging effect, skin wrinkle improvement effect, skin whitening effect, and skin damage inhibitory effect through the antioxidant effect of meadowsweet spirea extract, but studies on other uses has not been announced

The present invention discloses the anti-obesity use of bridal spirea extract.

An object of the present invention is to provide an anti-obesity composition using spirea extract.

Other objects or specific objects of the present invention will be presented below.

As confirmed in the following Examples and Experimental Examples, the present invention suppresses the differentiation of 3T3-L1, a mouse preadipocyte, into adipocytes in a concentration-dependent manner, and clearly inhibits weight gain in an obese animal model. , It was completed by confirming that it not only significantly reduced the amount of fat in visceral fat and subcutaneous fat, but also reduced the accumulation of liver fat, subcutaneous fat, epididymal fat, and subperitoneal fat, and reduced blood neutral fat.

Considering the experimental results as described above, the anti-obesity composition of the present invention is characterized in that it contains meadowsweet spirea extract as an active ingredient.

In the present specification, "spirea spirea extract" refers to bridal spirea leaves, stems, aerial parts, rhizomes, roots, underground parts or mixtures thereof to be extracted by water, lower alcohols having 1 to 4 carbon atoms (methanol, ethanol, butanol, etc.), methylene chloride , ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,3-butylene glycol, propylene glycol, or a mixed solvent thereof It refers to an extract obtained by leaching using an extract obtained by leaching, an extract obtained by using a supercritical extraction solvent such as carbon dioxide or pentane, or a fraction obtained by fractionating the extract. , reflux, heating, ultrasonic radiation, supercritical extraction, etc. may be applied. In the case of the fractionated extract, after suspending the extract in a specific solvent, the fraction obtained by mixing and standing with a solvent of different polarity, and the crude extract are adsorbed on a column filled with silica gel, etc., and then a hydrophobic solvent, a hydrophilic solvent, or a mixed solvent thereof It is meant to include fractions obtained as a mobile phase. In addition, the meaning of the extract includes a concentrated liquid extract or solid extract from which the extraction solvent is removed by lyophilization, vacuum drying, hot air drying, spray drying, or the like. Preferably, it refers to an extract obtained by using water, an alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof as an extraction solvent, and more preferably an extract obtained by using a mixed solvent of water and an alcohol having 1 to 4 carbon atoms as an extraction solvent. do.

In addition, in the present specification, "active ingredient" means a component that exhibits the desired activity alone or can exhibit activity in combination with a carrier having no activity itself.

Also, in the present specification, "anti-obesity" means a decrease in body fat, inhibition of body fat accumulation, and/or a decrease in body weight. Therefore, it means to include prevention of obesity and treatment of obesity, and further includes reducing body weight/body fat for beauty or health purposes (aka diet purposes) although weight is not classified as obesity or overweight.

In addition, in the present specification, the "obesity" refers to a state in which adipose tissue is abnormally increased, whether it is obesity caused by genetic factors or obesity caused by environmental factors, and according to the classification of body mass index (BMI), high This includes obesity (BMI greater than 30.0), obesity (BMI between 25 and 30), and overweight (BMI between 23 and 25).

The anti-obesity composition of the present invention may be included in any amount (effective amount) according to use, formulation, blending purpose, etc., as long as the active ingredient can exhibit anti-obesity activity. A typical effective amount is based on the total weight of the composition It will be determined within the range of 0.001% by weight to 20.0% by weight when Here, "effective amount" means that when the composition of the present invention is administered to a mammal, preferably a human, to which it is applied, during the administration period according to the advice of medical experts, the intended functional and pharmacological effects such as anti-obesity effect can be exhibited, It refers to the amount of the active ingredient included in the composition of the present invention. Such an effective amount can be determined empirically within the ordinary skill of the skilled artisan.

In addition to the active ingredients, the anti-obesity composition of the present invention has already been proven safe in the art in order to enhance the convenience of intake or intake through the addition of similar activities such as blood pressure control activity or to increase or reinforce the anti-obesity effect. It may further include any compound or natural extract known to have that activity. These compounds or extracts include compounds or extracts, pharmaceuticals listed in compendials such as pharmacopoeia of each country ("Korean Pharmacopoeia" in Korea) and health functional food codes of each country (in Korea, it is "standards and specifications for health functional foods", a notice of the Ministry of Food and Drug Safety). The laws of each country regulating the manufacture and sale of compounds or extracts and health functional foods that have been licensed under each country's laws ("Pharmaceutical Affairs Act" in Korea) governing the manufacture and sale of health functional foods ("Health Functional Foods Act" in Korea) ”), compounds or extracts whose functionality has been recognized are included.

For example, Garcinia cambogia peel extract, conjugated linolenic acid (free fatty acid), conjugated linolenic acid (triglyceride), green tea extract, chitosan, Lactobacillus gasseri, whose functionality has been recognized for 'body fat reduction' according to the "Health Functional Food Act" of Korea Complex such as BNR17 ( Lactobacillus gasseri BNR17), L-carnitine tartrate, green mate extract, green coffee bean extract, sesame leaf extract, soybean embryo extract, rhododendron leaf alcohol extract powder, lactoferrin (milk purified protein), lemon balm extract mixture powder , Mate hot water extract, seaweed and other complex extracts (Xanthigen), fermented vinegar pomegranate complex, Puer tea extract, Seomoktae (swine pea) peptide complex, vegetable oil diglyceride, wild mango seed extract, medium chain fatty acid (MCFA)-containing oil, Coleus forskoli extract, chitooligosaccharide, fingerroot extract powder, complex extracts such as hibiscus, etc., L-glutamic acid-derived GABA-containing powder whose functionality has been recognized as 'blood pressure control', katsuobushi oligopeptide, Natto bacteria cultured powder, Seomoktae Soybean) Peptide complex, salmon peptide, olive leaf extract, sardine peptide, casein hydrolysate, coenzyme Q10, grape seed enzyme decomposition extract powder, Haitai oligopeptide, etc., DHA concentrated oil and globin hydrolyzate recognized as 'improving blood triglycerides' , indigestible maltodextrin, bamboo leaf extract, vegetable oil diglyceride, sardine-refined fish oil, refined squid oil, etc., L-arabinose whose functionality has been recognized as 'glycemic control', nopal extract, cinnamon extract powder, guava leaf extract, ovary Hwaseong maltodextrin, freeze-dried silkworm powder, marijuana extract, banaba leaf extract, leaf extract, etc., fermentation-produced amino acid complex whose functionality has been recognized as 'fatigue improvement', hovenia pedunculus extract, rhodiola extract, etc., functional as 'anti-stress' These recognized L-theanine, ashwagandha extract, hydrolysate of milk protein, and extract of the leaves of the plant will correspond to these compounds or extracts.

In another aspect, the present invention relates to a composition for improving hyperlipidemia comprising bridal spirea extract as an active ingredient.

In the present specification, "hyperlipidemia" refers to a condition in which fat metabolism in the blood, such as triglyceride and cholesterol, is not properly performed, and the amount of fat in the blood is increased more than necessary, and is meant to include hypertriglyceridemia and hypercholesterolemia.

Also, in the present specification, "improvement of hyperlipidemia" means lowering the concentration of blood triglycerides or lowering the blood cholesterol concentration.

In the antihyperlipidemic composition of the present invention, the meaning of the spirea extract and the effective amount thereof are as described above in relation to the anti-obesity composition of the present invention.

The composition for anti-obesity and the composition for improving hyperlipidemia of the present invention can be identified as a food composition in a specific embodiment.

The food composition of the present invention can be prepared in any form, for example, beverages such as tea, juice, carbonated beverages, and ionic beverages, processed oils such as milk and yogurt, chewing gum, rice cakes, traditional Korean snacks, bread, snacks, noodles, etc. It can be manufactured into health functional food preparations such as foods, tablets, capsules, pills, granules, liquid, powder, flakes, pastes, syrups, gels, jellies, and bars.

In addition, the food composition of the present invention may take any product classification as long as it conforms to the enforcement regulations at the time of manufacture and distribution in legal and functional classification. For example, health functional food according to the Korean 「Health Functional Food Act」, or snacks, legumes, teas, beverages according to each food type according to the Food Code (MFDS notification 「Food Standards and Specifications」) of the Korea 「Food Sanitation Act」 , special purpose foods, etc.

The food composition of the present invention may include food additives in addition to the active ingredient. Food additives may generally be understood as substances that are added to, mixed with, or infiltrated into food in manufacturing, processing, or preserving food. Since food is consumed daily and for a long period of time, its safety must be guaranteed. According to the Food Additives Code under the laws of each country governing the manufacture and distribution of food (in Korea, it is the 「Food Sanitation Act」), safety-guaranteed food additives are limitedly regulated in terms of ingredients or functions. In the Korean Food Additives Codex (MFDS notification 「Standards and Specifications for Food Additives」), food additives are classified into chemical synthetic products, natural additives and mixed preparations in terms of ingredients and are regulated. It is divided into additives, preservatives, emulsifiers, acidulants, and thickeners.

Sweeteners are used to impart appropriate sweetness to foods, and both natural and synthetic sweeteners can be used in the food composition of the present invention. Preferably, a natural sweetener is used, and examples of the natural sweetener include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose, and maltose.

Flavors are used to improve taste or aroma, and both natural and synthetic flavors may be used. Preferably, it is the case of using a natural one. In case of using natural ones, in addition to flavor, the purpose of enhancing nutrition can also be combined. As a natural flavoring agent, it may be obtained from apples, lemons, tangerines, grapes, strawberries, peaches, etc., or obtained from green tea leaves, roundworms, bamboo leaves, cinnamon, chrysanthemum leaves, jasmine, and the like. In addition, those obtained from ginseng (red ginseng), bamboo shoots, aloe vera, ginkgo, etc. can be used. Natural flavors can be liquid concentrates or solid extracts. In some cases, synthetic flavors may be used, and as synthetic flavors, esters, alcohols, aldehydes, terpenes, and the like may be used.

As preservatives, calcium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid), etc. may be used, and as emulsifiers, acacia gum, carboxymethylcellulose, xanthan gum, pectin etc. may be used, and as an acidulant, acidulic acid, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid, and the like may be used. Acidulants may be added to the food composition to have an appropriate acidity for the purpose of inhibiting the growth of microorganisms in addition to improving taste. As the thickening agent, a suspending agent, a sedimentation agent, a gel forming agent, a swelling agent, and the like may be used.

In addition to the food additives described above, the food composition of the present invention may include physiologically active substances or minerals that are known in the art and whose stability is guaranteed as food additives for the purpose of supplementing and reinforcing functionality and nutrition.

Examples of such physiologically active substances include catechins contained in green tea, vitamins such as vitamin B1, vitamin C, vitamin E, and vitamin B12, tocopherol, dibenzoylthiamine, and the like. Examples of minerals include calcium preparations such as calcium citrate and magnesium stearate. magnesium preparations such as the like, iron preparations such as iron citrate, chromium chloride, potassium iodine, selenium, germanium, vanadium, zinc, and the like.

The food composition of the present invention may include the above-mentioned food additives in an appropriate amount to achieve the purpose of addition according to the type of product.

Regarding other food additives that may be included in the food composition of the present invention, reference may be made to national food codes or food additive codes.

The composition of the present invention can be understood as a pharmaceutical composition in another specific aspect.

The pharmaceutical composition of the present invention may be formulated into an oral formulation or a parenteral formulation according to the route of administration by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. The route of administration herein may be any appropriate route including topical route, oral route, intravenous route, intramuscular route, and direct absorption through mucosal tissue, and two or more routes may be used in combination. An example of a combination of two or more routes is a case in which two or more formulations of drugs are combined according to the route of administration, for example, one drug is firstly administered intravenously and the other drug is secondly administered through a topical route.

Pharmaceutically acceptable carriers are well known in the art depending on the route of administration or dosage form, and specifically, reference may be made to the pharmacopoeia of each country including the "Korean Pharmacopoeia".

When the pharmaceutical composition of the present invention is prepared as an oral dosage form, powder, granule, tablet, pill, dragee, capsule, liquid, gel, syrup, suspension, wafer according to a method known in the art together with a suitable carrier. It can be prepared in formulations such as Examples of suitable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, and xylitol, starches such as corn starch, potato starch, and wheat starch, cellulose, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, Celluloses such as hydroxypropylmethylcellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable oil, ethanol, Serol etc. are mentioned. In the case of formulation, appropriate binders, lubricants, disintegrants, coloring agents, diluents, etc. may be included as needed. Suitable binders include starch, magnesium aluminum silicate, starch ferrite, gelatin, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, glucose, corn sweetener, sodium alginate, polyethylene glycol, wax, and the like, and oleic acid as a lubricant Sodium, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, magnesium salts and calcium salts thereof, polyethylene glycol, etc. may be mentioned. As disintegrants, starch, methyl cellulose , agar, bentonite, xanthan gum, starch, alginic acid or its sodium salt, and the like. Examples of the diluent include lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and glycine.

When the pharmaceutical composition of the present invention is prepared as a parenteral formulation, it may be formulated in the form of an injection, transdermal administration, nasal inhalation, and suppository along with a suitable carrier according to a method known in the art. When formulated as an injection, an aqueous isotonic solution or suspension may be used as a suitable carrier, and specifically, an isotonic solution such as phosphate buffered saline (PBS) containing triethanolamine, sterile water for injection, or 5% dextrose may be used. . When formulated as a transdermal formulation, it may be formulated in the form of ointments, creams, lotions, gels, external solutions, pastas, liniments, air rolls, and the like. In the case of nasal inhalation, it can be formulated in the form of an aerosol spray using a suitable propellant such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, etc., and when formulated as a suppository, the carrier is Witepsol ( witepsol), tween 61, polyethylene glycols, cacao fat, laurin fat, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, sorbitan fatty acid esters, and the like can be used.

Regarding the specific formulation of the pharmaceutical composition, it is known in the art, and for example, reference may be made to Remington's Pharmaceutical Sciences (19th ed., 1995) and the like. These documents are considered as part of this specification.

The preferred dosage of the pharmaceutical composition of the present invention is in the range of 0.001 mg/kg to 10 g/kg per day, preferably 0.001 mg/kg to 1 g, depending on the patient's condition, weight, sex, age, severity of the patient, and route of administration. /kg range. Administration can be done once a day or divided into several times. These dosages should not be construed as limiting the scope of the present invention in any respect.

As described above, according to the present invention, it is possible to provide an anti-obesity composition and an anti-hyperlipidemic composition using brisket extract. The anti-obesity composition of the present invention and the like can be commercialized as functional foods, medicines, and the like.

Figure 1 is a result of quantifying the fat amount of mouse preadipocytes, 3T3-L1, treated with brisket extract by concentration, and then stained with Oil Red O reagent and stained with a microscope.
Figure 2 is a configuration diagram of an obese animal model experiment.
Figure 3 is the result of measuring the body weight and feed intake.
4 is a result of measuring visceral fat and subcutaneous fat.
5 is a result of measuring blood neutral fat.
6 is a photograph of liver adipose tissue staining.
7 is a photograph of the size of adipocytes in liver tissue and a result of quantifying it as a graph.
8 is a result of measuring leptin concentration in blood.

Hereinafter, the present invention will be described with reference to Examples and Experimental Examples. However, the scope of the present invention is not limited to these Examples and Experimental Examples.

< Example > Preparation of meadowsweet extract

Meadowsweet (extracted parts, leaves) were washed with distilled water 2-3 times, dried, dried with hot air, and ground with a grinder. Thereafter, distilled water was added to the pulverized product at a ratio of 1:10 (w:v), stirred and extracted at 80 ° C. for 3 hours, the extract was filtered, and the filtrate was concentrated under reduced pressure and freeze-dried to obtain a meadowsweet extract.

< Experimental example > Obesity improvement activity test of meadowsweet spirea extract

<Experimental Example 1> Adipocyte differentiation inhibitory activity test

Mouse proadipocytes, 3T3-L1 (ATCC CL-173), were cultured in 10% BCS DMEM medium at 37°C and 5% CO ₂ . 3T3-L1 preadipocytes were dispensed in a 24 well plate at a cell count of 5×10 ⁴ /well, and then maintained for 2 days at 100% confluency. Preadipocytes were differentiated into adipocytes with 10% FBS DMEM medium containing MDI (0.5 mM 3-isobutyl-1-methylxanthine (Calbiochem 410957), 1 uM dexamethasone (Calbiochem 265005), 1 ug/ml insulin (Sigma I9278)) was induced for 2 days, and after 48 hours of culture, it was cultured for 2 days with 10% FBS DMEM containing 1 ug/ml insulin. After that, the culture medium was replaced with 10% FBS DMEM every 2 days for 4 days. During the induction of differentiation into adipocytes, the extracts of Examples were treated with the respective cultures at concentrations of 50 and 100 μg/ml, and the degree of differentiation into adipocytes was observed on the 8th day when differentiation was completed. The degree of differentiation of adipocytes was first confirmed through a microscope through Oil Red O staining, and the degree of adipocyte staining was measured by using an ELISA Reader (SPECTRAmax 340PC, Molecular Devices, USA) at 510 nm absorbance to measure the amount of differentiated fat cells. did The results were calculated through repeated experiments three times for each concentration.

The results are shown in FIG. 1, and referring to FIG. 1, it can be seen that the meadowsweet extract has concentration-dependent activity of inhibiting differentiation of pre-adipocytes into adipocytes and inhibiting activity of fat accumulation.

<Experimental Example 2> Obesity animal model experiment

1. Experiment method

1.1 Production of obesity animal model by high fat diet

After purchasing 6-week-old C57BL/6 mice and arranging them in ascending order of weight, and separating groups (n = 10) so that the average and standard deviation between groups are equal, as shown in the table below and FIG. 2, the control group (HFD) A high-fat diet (containing 60% fat) was fed, and the experimental groups (50, 100, 200, 400 MPK) were fed with the high-fat diet and the sample of the above example. The high-fat diet was freely consumed, and the example samples were orally administered at concentrations of 50, 100, 200, and 400 mg/kg for 6 weeks using a sonde every day. The sample was not administered to the normal group (ND) and the normal feed was fed.

1.2 Measurement of changes in body weight and food intake

During the six-week experiment, body weight and feed intake were measured twice a week.

1.3 Visceral fat, subcutaneous fat imaging, fat mass measurement

At the 3rd and 6th weeks of the 6-week experimental period, all animals were imaged using animal micro-CT (Inveon, Simens). The amount of visceral fat and subcutaneous fat of animals was quantitatively analyzed using the photographed image files.

1.4 Quantitative analysis of blood triglycerides

After oral administration for 6 weeks, animals were sacrificed and blood was collected from the abdominal aorta. The collected blood was centrifuged at 3,000 rpm using a centrifuge for analysis, and plasma was separated, and blood triglyceride levels were measured using a blood biochemical analyzer (Hitachi 7020, Japan).

1.5 Adipose tissue staining for each part, such as the liver, measuring the amount of change in adipose tissue and the size of fat tax

After sacrificing the experimental animals, liver, subcutaneous fat, epididymal fat, and peritoneal fat were extracted for each part. Thereafter, after fixing with 4% formaldehyde for 7 days, the fixed tissue was further subdivided into 1 × 0.5 cm in size, placed in a tissue cassette, and used in a tissue processor (Thermo scientific, USA). After going through dehydration, transparency, and paraffinization, and storing at -20℃, cut the paraffin block into a thickness of 4~5㎛ with a microtome (Thermo scientific, USA) and dry it on a heating plate at 60℃. made it To measure skin tissue lesions, sections of skin tissue were deparaffinized with xylene, and then the deparaffinized skin tissue was treated with hematoxylin/eosin, toludin blue O solution ( toluidine blue O solution) to stain each tissue cell, and after encapsulation using a mount regent, it was observed under an optical microscope. Next, the adipose tissue slides stained with toluidine blue O were compared and analyzed by measuring the size of adipose tissue for each site with an optical microscope and calculating averages and percentages.

1.6 Blood Leptin Assay

Leptin is a type of adipokine that increases insulin sensitivity in muscle and liver and affects glucose utilization in peripheral blood. Blood concentration is increased due to leptin resistance in obese states (Nature 1998;392:398 -401). Therefore, substances exhibiting anti-obesity activity reduce the concentration of leptin in the blood.

In this experiment, blood leptin was measured using ELISA. Specifically, the anti-leptin/adiponectin coated plate was treated by reacting the primary antibody, biotin-conjugated anti-leptin, with plasma for 2 hours, , After washing three times, a secondary antibody, HRP-conjugated streptavidin, was added and reacted for 1 hour. After washing 4 times, the substrate was added, reacted for 30 minutes, and the reaction was stopped by adding a stop buffer, and absorbance was measured at 450 nm with an ELISA reader. Leptin was quantified in comparison with the standard curve.

2. Experimental results

1.1 Results of measuring changes in body weight and food intake

During the six-week experiment, body weight and feed intake were measured twice a week, and the results are shown in FIG. Body weight increased the most in the control group, and the degree of increase was significantly lower in the experimental group than in the control group. In Table 2 below, the degree of weight suppression in the experimental group compared to the control group for 6 weeks was shown as a percentage. The amount of food did not show any significant difference between the control group and the experimental group.

1.2 Results of visceral fat, subcutaneous fat imaging, and fat mass measurement

The results of measuring visceral fat and subcutaneous fat during the 6-week test period are shown in FIG. 4 . Both visceral fat and subcutaneous fat decreased significantly in proportion to the dose in the experimental group compared to the control group. The lower picture of FIG. 4 is a picture of visceral fat.

1.3 Quantitative analysis of blood triglycerides

The results of blood triglyceride measurement during the 6-week test period are shown in FIG. 5 . Referring to FIG. 5 , blood triglycerides were markedly lower in all experimental groups than in the control group.

1.4 Results of adipose tissue staining, adipose tissue change, and fat tax size measurement for each part, such as the liver

The fat accumulation inhibition rate and body fat percentage of subcutaneous fat, epididymal fat, and subperitoneal fat compared to the control group are shown in Tables 3 to 5 below, respectively. It can be seen that subcutaneous fat, epididymal fat, and subperitoneal fat all decreased in proportion to the dose.

In addition, a picture of liver adipose tissue staining is shown in FIG. 6 . Even in liver tissue, it shows that adipose tissue decreases in proportion to the dose in the experimental group compared to the control group.

In addition, a photograph of the size of adipocytes in liver tissue and a result of quantifying it in a graph are shown in FIG. 7 . It shows that the size of adipocytes also decreased in proportion to the dose in the experimental group compared to the control group.

1.5 Blood Leptin Assay

Results are shown in FIG. 8 . Figure 8 shows that the concentration of leptin in the blood decreased in proportion to the dose in the experimental group compared to the control group.

Claims (8)

Meadowsweet ( Spiraea prunifolia ) A functional food composition for anti-obesity containing a hot water extract of leaves as an active ingredient.
delete Meadowsweet ( Spiraea prunifolia ) Anti-obesity pharmaceutical composition comprising a hot water extract of leaves as an active ingredient.
delete A functional food composition for improving hyperlipidemia, comprising a hot water extract of Spiraea prunifolia leaves as an active ingredient.
delete A pharmaceutical composition for improving hyperlipidemia, comprising a hot water extract of Spiraea prunifolia leaves as an active ingredient.

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