KR101615026B1 - Compositions for preventing or treatmenting obesity or lipid-related metabolic disease comprising syringic acid, or salt thereof as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for preventing, ameliorating or treating obesity or lipid-related metabolic diseases comprising sialic acid, or a pharmaceutically or pharmacologically acceptable salt thereof as an active ingredient. The composition reduces weight gain, lipid content in blood and liver tissues of obese-induced individuals, promotes expression of fatty acid oxidation-related genes, and inhibits expression of lipid synthesis-related genes and enzymes. It also inhibits the activity of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), decreases leptin and insulin resistance, and increases adiponectin content. It also inhibits the expression of inflammatory factors and inflammatory genes in liver and increases the expression of antioxidant metabolism-related enzymes and lipid oxidation-related proteins, thereby preventing, ameliorating, or treating obesity or lipid-related metabolic diseases The composition of the present invention can be used as a composition which can be used. In addition, since the Sering acid is a chemical substance derived from a plant, the side effects on the human body are extremely lower than the chemical synthesis, so that it can be safely applied to pharmaceutical and food compositions.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing or treating obesity or a lipid-related metabolic disease comprising sialic acid or a salt thereof as an active ingredient,

The present invention relates to a composition for the prevention or treatment of obesity or lipid-related metabolic diseases, and more particularly to a composition for preventing or treating obesity or lipid-related metabolic diseases. More particularly, the present invention relates to a composition for preventing or treating obesity or lipid- ), Lipid metabolism-related enzymes and gene expression regulation, lipid-related metabolic diseases caused by obesity or obesity, including sialic acid, or pharmaceutically or pharmacologically acceptable salts thereof, which regulate inflammatory factors and inflammation-related gene expression Prevention or treatment of cancer.

In recent years, with the economic growth in Korea, the westernization of dietary habits has increased the calories consumed and the obesity population is increasing accordingly. Specifically, according to the Health and Welfare Statistical Yearbook of 2013, the proportion of obese people aged 19 and over is 31.4%, which is 0.5% higher than the 30.9% surveyed in 2010, and the rate of childhood obesity is also rapidly increasing.

Obesity itself increases the body weight and increases the size of the body, which makes it inconvenient for life, but the bigger problem is the increase of the lipid concentration in the blood, the arteriosclerosis and the heart disease, and the insulin resistance increases the diabetes, menstrual impurities, It is essential to treat and prevent obesity because it causes complications and causes chronic adult diseases such as hyperlipemia, hypertension, coronary artery and stroke (Lee JH, J. Kor. Soc. Obes., 1: 21-24, 1992. Lew EA, Ann. Med., 103: 1024-1029, 1985; Kim KI et al ., J. J. Food Sci. Technol., 35: 720-725, 2003).

The causes of obesity are known to be caused by genetic influences, environmental effects of westernized eating habits, and psychological effects due to stress, but the precise cause and mechanism of obesity is unclear.

In the past, adipocytes have simply been recognized as cells that store the surplus energy of the human body in the form of triglycerides and act as shock cushions from the outside. Recently, however, adipocytes are recognized as endocrine organs that secrete adipocytokines that regulate fasting, metabolism, and insulin sensitivity. Specifically, adipocytes such as adiponectin, leptin, resistin, tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6) Adipocytokines are known to play an important role in homeostasis and energy metabolism regulation (Matsuzawa, Y. et al. , Ann. N. Acad Sci., 892: 146-154, 1999; Med., 7: 887-888, 2001).

In particular, leptin produced by obesity genes is proportional to the amount of adipocytes. Therefore, the concentration of serum leptin in obese persons is higher than that of normal weight, and more is produced in subcutaneous fat than visceral fat. It plays an important role in obesity, not only in controlling eating, but also in energy consumption and reproductive function, suppressing appetite and increasing heat production through the sympathetic nervous system. In addition, circulating the brain to act on the hypothalamus receptors to suppress the diet, and increased body fat as a secreted substance in the brain as a satiety center to control the satiety center to stimulate the brain to inhibit appetite (Mantzoros, CS, Ann Intern. Med., 130: 671-680, 1999; Roemmich, JN and AD, Am. J. Hum. Biol., 11: 209-224, 1999).

There are two main goals to treat obesity. The first is to burn excess fat to reduce body weight, and the second is to improve metabolic imbalance. Currently, obesity treatment aims not only to lose weight, but also to improve metabolic abnormalities by eliminating factors that cause early cardiovascular disease. In addition, studies to suppress obesity through controlling dietary intake and energy consumption have been actively conducted. The hypothalamus, especially the hypothalamus, plays an important role in the pathogenesis of obesity. Neuropeptide Y, POMC / CART, melanocyte, melanocyte, Cholinesterase, norepinephrine, serotonin, etc. are secreted from the hypothalamus. Current strategies for the development of obesity therapies are such as reducing food intake, inhibiting the absorption of heat, promoting exothermic reactions, regulating energy metabolism, and regulating signaling through the nervous system (MJ Jung, J Pediatr 48 (2), 2005).

The most commonly known drugs for obesity are classified as fullness supple- ments, fat absorption inhibitors and antipsychotic appetite suppressants depending on the mechanism of action. The most popular drugs are Xenical ^TM (Roche Pharmaceuticals, Switzerland), Reductil ^TM (Abbott, USA) ), Exorise (Exolise ^TM , Atofarma, France), but it also causes side effects such as diarrhea, intestinal gas, abdominal bloating, defecation incontinence, heart disease, respiratory diseases and neurological diseases. There is a problem that the sustainability is low.

In order to minimize the side effects of artificially synthesized materials, functional materials effective for controlling body weight from natural materials have been developed. Representative examples include green tea, phosphoric acid, Angelica gigas, Shiga mushroom, red ginseng, Mulberry tree, and brown algae. However, in the case of the anti-obesity substance extracted from such a natural substance, there is a problem that the effective concentration of the substance showing efficacy is low, and it is necessary to cultivate it in agricultural land or the like.

Accordingly, the present inventors have made efforts to develop a composition capable of preventing, ameliorating or treating obesity or a lipid-related metabolic disease in a phytochemical derived from a plant extract. As a result, the present inventors have found that the use of Sialic acid or a pharmaceutically or pharmacologically acceptable salt thereof The composition comprising the active ingredient may be used to reduce the body weight gain, the lipid content in blood and liver tissue of an individual who induces obesity, to control the expression of adipocytokine, to control lipid metabolism-related enzyme and gene expression, And a method for preventing, ameliorating or treating obesity or lipid-related metabolic diseases by controlling expression of an inflammatory metabolism-related gene. The present invention has been completed based on this discovery.

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating obesity, which comprises sialic acid or a pharmaceutically acceptable salt thereof as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of lipid-related metabolic diseases comprising sialic acid or a pharmaceutically acceptable salt thereof as an active ingredient.

It is still another object of the present invention to provide a food composition for preventing or improving obesity, which comprises sicric acid or a pharmaceutically acceptable salt thereof as an active ingredient.

It is still another object of the present invention to provide a food composition for preventing or ameliorating a lipid-related metabolic disease comprising sialic acid or a pharmaceutically acceptable salt thereof as an active ingredient.

In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating obesity or lipid-related metabolic diseases comprising sialic acid or a pharmaceutically acceptable salt thereof as an active ingredient.

In the present invention, the syringic acid is one of the phenolic compounds. The IUPAC name is 4-hydroxy-3,5-dimethoxybenzoic acid, Structure.

The syringic acid is a phenolic compound synthesized by the shikimic acid pathway and can be extracted, separated and purified from plant species such as sorghyum bicolor and Euterpe oleracea , (For example, syringic acid from Sigma-aldrich). It is known that the Sering acid exhibits the effects of antioxidation, whitening activity, antibacterial and anticancer effects (Korean Patent No. 10-1283849; Journal of agricultural science, Khim PC, 2009, 1 (2): 15-20 .; J Exp. Biomed. Sci., Lee et al. , 2005. 11: 337-341.). However, the preventive, ameliorative or therapeutic efficacy of obesity and / or lipid-related metabolic diseases in Sichuan is still unknown.

In the case of extracting the sialic acid from the plant, it can be extracted using water or an organic solvent. The extracted liquid can be used in a liquid form or can be used by concentration and / or drying. The organic solvent may include, but is not limited to, methanol, ethanol, isopropanol, butanol, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, dichloromethane, N, N-dimethylformamide (DMF) (DMSO), 1,3-butylene glycol, propylene glycol or a mixed solvent thereof, preferably methanol, ethanol, butanol, hexane, dichloromethane, ethyl acetate or a mixed solvent thereof, Can be extracted at room temperature or warming under non-destructive or minimized conditions. Depending on the organic solvent to be extracted, the degree of extraction and the degree of loss of the active ingredient of the extract may differ. Therefore, an appropriate organic solvent should be selected and used. The extraction method is not particularly limited, and examples thereof include cold extraction, ultrasonic extraction, and reflux cooling extraction.

In addition, the above-mentioned seeding acid can be obtained by a conventional purification process in addition to the extraction method using the above-mentioned extraction solvent. For example, by separation using an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatographies (made for separation by size, charge, hydrophobicity or affinity), and the like, Sulfuric acid can also be obtained through fractionation.

In the present invention, the pharmaceutically acceptable salt of the above-mentioned sialic acid is useful as an acid addition salt formed by a pharmaceutically acceptable free acid or a metal salt formed by a base. As an example, inorganic acid and organic acid can be used as the free acid. As the inorganic acid, hydrochloric acid, sulfuric acid, bromic acid, sulfurous acid or phosphoric acid can be used. As the organic acid, citric acid, acetic acid, maleic acid, fumaric acid, gluconic acid, methanesulfonic acid and the like can be used. As the metal salt, an alkali metal salt or an alkaline earth metal salt, sodium, potassium or calcium salt may be used. But is not necessarily limited thereto.

In the present invention, obesity means a condition or disease having excessive body fat due to energy imbalance.

In the present invention, a lipid-related metabolic disease refers to a disease caused by excessive lipid accumulation in vivo. Specific examples thereof include at least one selected from the group consisting of diabetes, hyperlipidemia, fatty liver, hepatitis, liver cirrhosis, arteriosclerosis, hypertension, cardiovascular disease, and metabolic syndrome in which the above diseases occur simultaneously.

The pharmaceutical composition of the present invention reduces weight gain, body fat mass, expression of gene related to lipogenesis and activity of lipogenesis-related enzyme of obesity-induced individuals by the active ingredient sialic acid or its pharmaceutically acceptable salt, Promotes fatty acid oxidation, and has an effect of preventing or treating obesity or lipid-related metabolic diseases by controlling the expression of adipocytokines, inflammatory factors, antioxidant metabolism-related enzymes, and lipid oxidation-related proteins.

In one specific embodiment, when sialic acid was fed to obesity-inducing experimental animals, body weight was significantly reduced without changes in dietary intake and diet efficiency compared to the obesity-induced experimental animals (high fat control group) (Triglyceride) content in the liver was decreased by 44% and the triglyceride content in the serum was decreased by 12%. In addition, the expression of AdipoR2, PPARα, ACSL, CPT1 and CPT2, which are involved in fatty acid oxidation, in the obese-induced experimental animals (high fat control group) was 118% The activities of beta-oxidation and carnitine acyl transferase (CPT) were increased by 106% and 47%, respectively. On the other hand, the expression of CIDEA, PPARγ, SREBF and FASN, which are genes related to lipid synthesis, decreased by 72%, 40%, 37% and 43%, respectively, and that of lipid synthesizing enzymes (phosphatidylserine phosphatase (PAP) FAS) activity was reduced by 33% and 61%, respectively.

In another specific embodiment, when sialic acid is fed to obesity-inducing experimental animals, asparate aminotransferase (AST) and alanine aminotransferase (AST), which are hepatotoxic enzymes, The activity of alanine aminotransferase (ALT) was decreased, the concentration of adiponectin was increased by 17%, and the concentration of leptin was decreased by 34%. In addition, when sialic acid was fed to obesity-induced experimental animals, insulin resistance was reduced by 63% as compared to obesity-induced experimental animals (high fat control group).

In another specific example, when sialic acid was fed to obesity-inducing experimental animals, TNF-α, INF-γ and MCP-1, which are inflammatory factors in serum, were significantly higher than those of obesity-induced experimental animals (high fat control group) The expression of inflammatory genes TLR4, MYD88, NF-κB, TNF-α and IL-6 was 58%, 71%, 65%, 76% and 69%, respectively. % Inhibition. The activity of CAT, GSH-Px and GST, which are antioxidative metabolism related enzymes, was 48%, 40% and 29%, respectively, in obesity-induced experimental animals (high fat control group) %, And the content of PPARα, a lipid metabolism related protein, was increased by 15%.

As used herein, the term "prophylactic " means inhibiting the occurrence of a disease or disorder in an individual who has never been diagnosed as having a disease or condition, but is likely to be susceptible to such disease or disease. As used herein, the term " improvement "or" treatment "means (a) inhibition of the development of a disease or disease in an individual, (b) relief of the disease or disorder, and (c) As used herein, the term "individual" means a mammal, including a human, having a disease whose symptoms may be ameliorated by administration of the composition of the present invention, such as a monkey, cow, horse, pig, sheep, dog, cat, rat, Means a mammal.

In the present invention, the pharmaceutical composition may additionally contain ingredients conventionally used in pharmaceutical compositions in addition to the above-mentioned sialic acid or a pharmaceutically acceptable salt thereof. For example, lubricants, wetting agents, flavoring agents, emulsifying agents, suspending agents, preservatives and carriers.

Carriers such as lactose, amylose, dextrose, sucrose, sorbitol, mannitol, starch, cellulose, etc., acacia rubber, calcium phosphate, Alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, salt solution, alcohol, gum arabic, vegetable oil such as corn oil, cottonseed oil, soybean oil, olive oil, coconut oil ), Polyethylene glycol, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition may be administered to mammals such as rats, mice, livestock, humans, and the like in various routes such as oral, parenteral, and the like, and all manner of administration may be expected, for example oral, rectal or intravenous Intraperitoneal, intramuscular, subcutaneous, or topical administration.

Suitable dosages of the pharmaceutical composition may be varied depending on factors such as the formulation method, the mode of administration, the patient's age, weight, sex, pathology, food, time of administration, route of administration, excretion rate and responsiveness have. On the other hand, the dosage of the pharmaceutical composition of the present invention is preferably 0.1-100 mg / kg (body weight) per day. When the composition is an external preparation, it is preferable to apply the composition in an amount of 1.0 to 3.0 mL on an adult basis once to five times a day for one month or longer. However, the dose is not intended to limit the scope of the present invention.

The pharmaceutical composition may be prepared in the form of a unit dose by formulating it with a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by those having ordinary skill in the art to which the present invention belongs. Into a multi-dose container. The formulations may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of excipients, powders, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

The sialic acid or a pharmaceutically acceptable salt thereof may be contained in an amount of 0.0001 to 10% by weight, preferably 0.0001 to 5% by weight, based on the total weight of the pharmaceutical composition. When the content is less than 0.0001% by weight, the effect of preventing or treating obesity and / or lipid-related metabolic diseases is too weak. When the content is more than 10% by weight, There is a problem that it can not be secured.

In another aspect, the present invention relates to a food composition for preventing or ameliorating an obesity or lipid-related metabolic disease comprising sialic acid or a pharmaceutically acceptable salt thereof as an active ingredient.

In the present invention, the structure, extraction method, efficacy, use and the like of the above-mentioned sulfic acid are as described above.

In the present invention, the pharmacologically acceptable salt of the above-mentioned sialic acid is useful as an acid addition salt formed by a pharmaceutically acceptable free acid or a metal salt formed by a base. As an example, inorganic acid and organic acid can be used as the free acid. As the inorganic acid, hydrochloric acid, sulfuric acid, bromic acid, sulfurous acid or phosphoric acid can be used. As the organic acid, citric acid, acetic acid, maleic acid, fumaric acid, gluconic acid, methanesulfonic acid and the like can be used. As the metal salt, an alkali metal salt or an alkaline earth metal salt, sodium, potassium or calcium salt may be used. But is not necessarily limited thereto.

The food composition according to the present invention may contain components that are conventionally added in the manufacture of food, in addition to the syringic acid or a pharmaceutically acceptable salt thereof. For example, proteins, carbohydrates, fats, nutrients, flavoring agents, and flavoring agents. The above-mentioned carbohydrates are polysaccharides such as disaccharides such as monosaccharide, maltose, sucrose and oligosaccharides of glucose and fructose sugars, dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. The flavoring agent may be a natural flavoring agent (tau martin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavor (saccharin, aspartame, etc.).

There is no particular limitation on the kind of the food. Examples of the food to which the above-mentioned sialic acid or a pharmaceutically acceptable salt thereof can be added include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, Various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, all of which include health foods in a conventional sense.

When the food of the present invention is a beverage, it may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. The above natural carbohydrates may include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and natural sweeteners such as dextrin and cyclodextrin, and synthetic sweeteners such as saccharin and aspartame.

The food may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, A carbonating agent used in a carbonated beverage, and the like. In addition, the health food of the present invention may contain natural fruit juice, pulp for the production of beverages and vegetable drinks. These components may be used independently or in combination.

The sialic acid or a pharmaceutically acceptable salt thereof may be contained in an amount of 0.0001 to 10% by weight, preferably 0.0001 to 5% by weight, based on the total amount of the composition. When the content is less than 0.0001% by weight, the effect of preventing or ameliorating obesity and / or lipid-related metabolic diseases is too weak. When the content is more than 10% by weight, There is a problem that it can not be secured.

As described above, the pharmaceutical or food composition of the present invention is useful for reducing weight gain, reducing lipid content in blood and liver tissues, inhibiting lipid synthesis-related genes and enzyme expression, promoting expression of fatty acid oxidation-related genes, (AST) and alanine aminotransferase (ALT) activity, increase of adiponectin content, decrease of leptin content, decrease of insulin resistance, suppression of serum factor and inflammation related gene expression and antioxidant metabolism related enzymes and lipid oxidation protein , Thereby exhibiting an efficacy for preventing, ameliorating or treating obesity or lipid-related metabolic diseases. Sialic acid or its pharmaceutically acceptable or pharmacologically acceptable salt as an active ingredient in the composition of the present invention is a chemical substance derived from a plant and is very safe because the side effects on the human body are extremely small compared to chemical synthetic substances .

The SINGING acid of the present invention reduces body weight gain, lipid content in blood and liver tissues of obese-induced individuals, promotes expression of fatty acid oxidation-related genes, and inhibits the expression of lipid synthesis-related genes and enzymes. It also inhibits the activity of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), decreases leptin and insulin resistance, and increases adiponectin content. In addition, since it exhibits an effect of suppressing the expression of inflammatory factors and inflammation-related genes in serum, and increasing the expression of antioxidant metabolism-related enzymes and lipid oxidation-related proteins, it is useful as a composition capable of preventing or treating obesity or lipid-related metabolic diseases Can be used. In addition, since the Sering acid is a chemical substance derived from a plant, the side effects on the human body are extremely lower than the chemical synthesis, so that it can be safely applied to pharmaceutical and food compositions.

FIG. 1 shows the results of measurement of body weight and body weight gain of an experimental animal taking a high fat diet containing a general diet, a high fat diet, and a herbicide.
FIG. 2 shows the results of observation of the size and morphological changes of visceral adipose tissue of an experimental animal that consumed a high-fat diet containing a general diet, a high fat diet, and a seasoning acid using an optical microscope.
FIG. 3 shows the results of observing the degree of fat accumulation in the liver tissues of an experimental animal taking a high fat diet containing a general diet, a high fat diet, and a herbicide using an optical microscope.
FIG. 4 shows expression of adipoR2, PPARα, ACSL, CPT1, CTP2, CIDEA, PPARγ, SREBF and FASN in the liver tissues of the experimental animals fed the high fat diet containing the general diet, .
FIG. 5 is a graph showing the effect of lipid metabolism-related enzymes such as phosphatidic acid phosphatase (PAP), fatty acid synthetase (FAS) and glucose-lipid synthase (FAS) in liver tissues of experimental animals fed a high fat diet containing a general diet, Activity of 6-phosphate dehydrogenase (G6PD), beta-oxidation and carnitine acyltransferase (CPT).
FIG. 6 shows the results of fasting glucose, glucose tolerance, asparate aminotransferase (AST), alanine aminotransferase (alanine aminotransferase) and alanine aminotransferase (ELISA) using sera from laboratory animals fed a high fat diet containing the general diet, high fat diet, aminotransferase, ALT), insulin concentration, leptin concentration, adiponectin concentration, and insulin resistance (HOMA-IR).
FIG. 7 shows the results of measurement of the levels of TNF-.alpha., IFN-.gamma. And MCP-1, which are inflammatory cytokines in the serum of experimental animals fed a high fat diet containing the general diet, high fat diet and Siring acid.
FIG. 8 shows the expression of inflammation-related genes TLR4, MYD88, NF-κB, TNF-α and IL-6 in liver tissues of experimental animals fed a high-fat diet containing a general diet, Results.
FIG. 9 is a graph showing the antioxidative metabolism-related enzymes CYP2E1, SOD, CAT, GSH-Px, GST activity, H ₂ O _2, and GSH in the liver tissues of the experimental animals fed the high fat diet containing the general diet, As a result of the measurement.
FIG. 10 shows the results of measurement of lipid metabolism-related proteins AMPK, p-AMPK, ACC, p-ACC and PPAR? In liver tissues of experimental animals fed a high fat diet containing a general diet, to be.

Hereinafter, the present invention will be described in more detail with reference to Production Examples and Examples. It is to be understood that both the foregoing preparations and examples are for the purpose of illustrating the present invention more specifically and that the scope of the present invention is not limited by these preparations and examples according to the gist of the present invention, It will be obvious to those who have knowledge of.

Preparation Example 1 Preparation of Syringic Acid

Sicling acid was purchased from Sigma-Aldrich (USA).

Preparation Example 2: Breeding of experimental animals and sample preparation

2-1. Breeding of experimental animals

Twenty-four C57BL / 6J male rats aged 4 weeks old were purchased from the Jackson Laboratory, Jackson, USA, and were adapted to the general diet for 1 week, followed by 3 groups (normal group, high fat control group and high fat- The diets of Table 1 were divided and fed for 16 weeks. Specifically, all groups except for the normal group fed a high fat diet with 20% fat and 1% cholesterol for induction of obesity, and the normal diet fed general diet with 5% fat.

Animal breeding is carried out in individual polycarbonate cages (22 ± 2 ° C), humidity (50 ± 5%) and 12-hour intervals (08: 00 ~ 20: 00) polycarbonate cage).

(unit: %) Normal group High fat control group High Fat - Sichuan County Salary Group Choline bitartrate 0.2 0.2 0.2 _{D, L} -methionine ( _{D, L} -methionine) 0.3 0.3 0.3 AIN76-mineral mix 3.5 4.2 4.2 AIN76-vitamin mixture One 1.2 1.2 Casein 20 20 20 Corn oil 5 3 3 Pork oil (lard) - 17 17 Cholesterol - One One Cellulose 5 5 5 Sucrose 50 37 37 Corn starch 15 11.1 11.1 Syringic acid - - 0.05 Gross weight (%) 100 100 100 Tert-butylhydroquinone (tert-butylhydroquinone) 0.001 0.004 0.004

2-2. Serum preparation from laboratory animals

In Example 2-1, the rabbits were fasted for 12 hours and fasting blood was collected from the inferior vena cava. The collected blood was kept at room temperature for 30 minutes to allow the blood cells to settle, and the serum was separated by centrifugation at 3,000 rpm for 15 minutes using a centrifuge maintained at 4 ° C.

2-3. Long-term tissue collection of experimental animals

After harvesting the blood of the experimental animals in Example 2-2, the organ tissues (liver, kidney, heart, and adipose tissue) were immediately taken out and rinsed several times with PBS (phosphate buffered saline) And immediately quenched with liquid nitrogen and stored at -70 ° C.

Example 1: Measurement of change in body weight and dietary intake with the addition of sialic acid

During the breeding period, specificities such as the activity of the animals and the presence of behavioral abnormalities were observed and recorded. Body weight was measured every week, and dietary intake was measured daily during the rearing period. The body weight and dietary intake were expressed as the mean value per experimental group. A significant difference test was performed using Duncan's multiple range test (DMRT) using one-way ANOVA. The results are shown in FIG. 1 and Table 2.

Normal group High fat control group High Fat - Searing Sanjo Dietary intake (g / day) 4.09 ± 0.08 ^b 3.57 + 0.05 ^a 3.56 ± 0.05 ^a Dietary Intake Efficiency (%) 3.43 ± 0.08 ^a 4.79 ± 0.16 ^c 4.33 ± 0.07 ^b Mean ± standard error, ^abc Values with different superscripts between experimental groups indicate a significant difference at p <0.05 level using Duncan's multiple test.

As a result, weight gain was observed in all experimental groups during the experimental period, and the average weight gain was significantly higher in the high fat control group than in the normal control group, and the average weight increase in the high fat-sering group was decreased by 10% Which is similar to that of normal group.

Example 2: Measurement of visceral fat weight and morphological change according to salicylic acid

The fat weights of the abdomen, epididymis, kidney, and mesentery obtained in Preparation Example 2-3 were measured and the size and morphological changes of epididymal adipose tissue were observed using an optical microscope. The weight and size of the visceral adipose tissue were expressed as the mean value per experimental group, and significant difference test was performed by Duncan's multiple range test (DMRT) using one-way ANOVA. The results are shown in FIG. 2 and Table 3.

(Unit: mg / g B.W.) Normal group High fat control group High Fat - Sichuan County Salary Group Epididymis 38.50 ± 0.91 ^a 54.92 + - 1.95 ^c 46.14 + - 3.04 ^b Abdominal fat 11.22 + - 0.57 13.57 + - 0.55 12.89 ± 1.11 Fat around the kidney 2.70 ± 0.36 ^a 4.16 ± 0.45 ^b 2.79 ± 0.30 ^a Mesenteric fat 14.21 ± 1.56 12.80 ± 1.07 12.28 ± 0.92 Total visceral fat 66.64 ± 2.40 ^a 85.46 ± 1.86 ^b 74.09 + 4.15 ^a Mean ± standard error, ^abc Values with different superscripts between experimental groups indicate a significant difference at p <0.05 level using Duncan's multiple test.

As a result, the visceral fat and the fat around the kidney were increased in the high fat control group, and the total visceral fat was significantly increased compared to the normal group. However, the high fat - saline supplementation group significantly reduced the increased epididymal fat and peripheral fat, reducing the total visceral fat content by 13% and the lipid increased by high fat diet by 25%.

Example 3 Measurement of Changes in Lipid Level of Body by Sicinic Acid Feeding

3-1. Measurement of lipid content in serum

The triglyceride content, total cholesterol content, free fatty acid content, HDL-cholesterol content, and HTR value in the serum of the experimental animals collected in Preparation Example 2-2 were measured.

Specifically, the content of triglycerides in serum was measured using a kit for measuring triglycerides (Asan Pharmaceutical, Korea) according to the principle of color development using the enzyme method of McGowan et al. (1983). Serum triglycerides are degraded by lipoprotein lipase (LPL) into glycerol and fatty acids. Among them, glycerol forms L-α-glycerophosphate by the action of ATP and glycerol kinase (GK), which reacts with O ₂ and glycerophospho-oxidase H ₂ O ₂ . The peroxidase and 4-amino-antipyrin were treated to develop a red color. The absorbance at 550 nm was measured and compared with the glycerol standard curve.

The total cholesterol content in the serum was determined using a kit for measurement (ASAN PHARMACEUTICAL CO., KOREA) using the enzyme method of Allain et al. (1974). Since serum cholesterol exists in two forms of ester cholesterol (CE) and free cholesterol (FC), ester cholesterol was converted to fatty acid and free cholesterol by cholesterol esterase in order to quantify all of them. Free cholesterol is converted to △ ⁴ -cholestenone by cholesterol oxidase, and this product and substrate H ₂ O ₂ are converted to peroxidase, phenol and 4-amino-antipyrine (4-amino-antipyrine) to produce a red color, and the absorbance at 500 nm was measured. Measurements were quantified relative to cholesterol standard solutions.

The content of free fatty acids was measured by using a non-esterified fatty acid (NEFA kit, Wako, Osaka, Japan) according to the principle of color development using the enzyme method. Acyl-CoA, AMP and pyrophosphoric acid were produced by the action of acyl coenzyme A (CoA) synthetase on serum free fatty acids, and acyl coenzyme A oxidase was added thereto To produce 2,3-trans-enoyl-CoA and H ₂ O ₂ . This peroxidase (peroxidase) and 4-amino-antipirin (4-amino-antipyrin) and N-ethyl-N- (2- hydr oxy-3-sulfo-oropyl) - after processing by the color red to m -toruidine The absorbance at 555 nm was measured and compared with the free fatty acid standard curve.

LDL and VLDL, including apo B in lipoprotein, are precipitated by the action of tungstic acid and magnesium cations when treated with 500 μg of sodium tungstate and 1 mg of magnesium chloride, taking 100 μl of serum HDL-cholesterol (Warnick, 1982). After centrifugation, the supernatant was subjected to a color reaction in the same manner as total cholesterol, and the absorbance at 500 nm was measured and compared with the cholesterol standard solution.

HTR values were calculated using Equation 1 using total cholesterol and HDL-cholesterol measurements.

[Experimental Equation 1]

HTR (%) = {HDL-cholesterol (mg / dL) / total cholesterol (mg / dL)} x 100

The triglyceride content, total cholesterol content, free fatty acid content, HDL-cholesterol content and HTR value in the serum were expressed as average values per experimental group, and significant difference tests were performed using Duncan's multiple range test using a one-way ANOVA, DMRT) was performed. The results are shown in Table 4.

Normal group High fat control group High Fat - Searing Sanjo Triglyceride (mg / dL) 55.80 ± 2.66 ^a 67.32 ± 2.66 ^b 59.34 ± 2.66 ^a Total cholesterol (mg / dL) 123.36 ± 6.96 ^a 150.04 ± 5.80 ^b 141.14 + - 4.64 ^b Free fatty acid (mmol / L) 1.00 + - 0.01 1.14 + 0.07 0.99 ± 0.01 HDL-cholesterol (mg / dL) 90.10 ± 9.67 95.51 + - 2.71 112.53 + - 7.73 HTR (%) 74.96 ± 2.67 ^b 62.77 + 2.81 ^a 80.79 ± 5.40 ^b Mean ± standard error, ^ab Differences between groups with different superscripts indicate a significant difference at p <0.05 using Duncan's multiple test.

As a result, triglyceride and total cholesterol contents in the serum of the high fat control group were significantly increased by about 21% and 22%, respectively, compared with the normal group, and the triglyceride and total cholesterol contents in the serum were increased by 12% 6%.

On the other hand, there was no decrease in HDL-cholesterol content with the addition of sialic acid, but the HDL-cholesterol ratio (HTR) to total cholesterol increased to a similar level to that of the normal group.

3-2. Measurement of lipid content in liver tissue and feces

Lipids were extracted from the liver tissues of the experimental animals obtained in Preparation Example 2-3 according to the method of Folch et al. (1957). The cholesterol and triglyceride contents in the tissues were measured and corrected by Omedeo et al. (1983) . Specifically, 0.1 g of liver tissue was minced, 2 mL of chloroform methanol (2: 1) solution was added to homogenize, and lipids were extracted at 25 ° C. for 3 hours. 200 μl of 1 M H ₂ SO ₄ was added to the mixture, and the mixture was centrifuged at 2,000 rpm for 20 minutes at room temperature. Then, 1 ml of the lipid layer was transferred, and 1 ml of a chloroform solution containing 1% Triton X-100 was added And then dried with nitrogen gas. Then, after adding distilled water and sonication, triglycerides, free fatty acids and cholesterol contents were measured in the same manner as in Example 3-1.

The lipid content of the feces was also measured by the method of Folch et al. (1957), and the cholesterol and triglyceride contents in the feces were measured and corrected by Omedeo et al. (1983). Specifically, after the feces of the experimental animals raised in Production Example 2-1 were dried, 10 mL of a chloroform methanol (2: 1) solution was added to 0.5 g of dried feces, homogenized and then leached for 24 hours. After centrifugation at 3,000 rpm for 10 minutes at 20 ° C, 7 mL was transferred to a new test tube and dried with nitrogen gas. The dried sample was mixed with 1 ml of chloroform methanol, and 200 μl of the mixture was transferred to a new test tube and dried again. After 1 ml of ethanol was added to the dried sample and sonication, the content of cholesterol and triglyceride was measured in the same manner as in Example 3-1.

The triglyceride content, cholesterol content and free fatty acid content in the liver tissues and feces were expressed as average values per experimental group and Duncan's multiple range test (DMRT) was performed using a one-way ANOVA . The results are shown in Table 5.

Normal group High fat control group High Fat - Searing Sanjo Liver tissue Triglyceride (mg / g) 12.40 ± 1.77 ^a 62.00 3.54 ^c 34.54 ± 2.66 ^b Cholesterol (mg / g) 0.77 ± 0.39 ^a 6.96 + - 0.39 ^c 3.87 ± 0.39 ^b Free fatty acids (mmol / g) 4.58 ± 0.09 ^a 6.51 + - 0.24 ^c 5.92 + 0.04 ^b Feces Triglyceride (mg / g) 37.20 ± 2.66 ^a 62.89 ± 5.31 ^b 56.69 + - 3.54 ^b Cholesterol (mg / g) 53.36 + 4.64 ^a 599.38 +/- 12.76 ^b 677.88 +/- 17.40 ^c Mean ± standard error, ^abc Values with different superscripts between experimental groups indicate a significant difference at p <0.05 level using Duncan's multiple test.

The contents of triglycerides, cholesterol and free fatty acids in the liver tissues of the high fat control group were significantly increased compared with those of the normal group, and the contents of triglyceride, cholesterol and free fatty acid in the liver tissues of the high fat control group 44%, 44% and 9%, respectively.

The fecal cholesterol content was increased by exacerbation of cholesterol by the addition of sicolic acid.

3-3. Observation of fat accumulation in liver tissue

Morphological observation of liver tissues was performed as follows to examine the degree of fat accumulation in the liver tissues of the experimental animals collected in Preparation Example 2-3. Specifically, the liver tissue was fixed in 10% formaldehyde solution for 24 hours, washed with water, dehydrated from 60% alcohol to a rising concentration and embedded in paraffin. The hepatic tissue was cross-cut to a thickness of 4 μm and stained with hematoxylin-eosin (H & E) Respectively. Oil Red O staining was performed by freezing the liver tissue, cutting it to a thickness of 4 μm, attaching it on a slide glass, drying it and dyeing it with Oil Red O dye. Masson Trichrome stain was fixed on a slide glass with 4 ㎛ thickness and fixed with bouin solution and trichrome stained. The stained liver tissue cells were observed under an optical microscope (200x magnification). The results are shown in Fig.

As a result of the experiment, it was observed that the liver tissues of the high fat control group had fat accumulation around the portal vein, and the fat accumulation and fibrosis degree were decreased in the high fat - sering acid group compared to the high fat control group.

Example 4: Measurement of lipid metabolism-related gene expression and enzyme activity change by feeding sialic acid

4-1. Measurement of lipid metabolism related gene expression changes by sialic acid

1 mL of trizol (TRIzol, Invitrogen, Grand Island, NY) was added to 0.1 g of the liver tissue of the experimental animals obtained in Preparation Example 2-3, and homogenized using a tissue lyzer (QIAGEN, Germany) And centrifuged at 3,250 x g and 4 ° C for 10 minutes. Then, the supernatant was taken, and 200 μl of chloroform was added thereto. The mixture was centrifuged at 3,250 × g at 4 ° C. for 20 minutes. The supernatant was again taken and the same amount of isopropanol was added. g, and centrifuged at 4 캜 to obtain RNA pellets. The RNA pellet was then washed twice with 75% ethanol, dried and dissolved in DEPC (diethyl pyrocarbonate) treated distilled water. And diluted to a concentration of 0.5 to 10 μg / μl, and stored at -70 ° C. until analysis.

For cDNA synthesis, first-strand cDNA was synthesized using the QuantiTect Reverse Transcription kit (QIAGEN, Germany). 1 μl of the previously isolated RNA was mixed with 2 μl of gDNA extraction solution (wipeout buffer), and the volume was adjusted to 14 μl with RNase-free distilled water. The reaction was performed at 42 ° C for 2 minutes and then cooled on ice. The cDNA was synthesized by reacting with 1 μl of reverse transcriptase, 4 μl of RT buffer and 1 μl of RT primer mix at 42 ° C for 15 minutes, followed by reaction at 95 ° C for 3 minutes. Reverse transcriptase ) Was inhibited. The resulting cDNA was diluted in RNase-free distilled water, stored at -20 ° C, and used as a template for RT-qPCT (real-time quantitative PCR). Expression of the target gene was examined by RT-qPCT using the obtained cDNA and RT-PCR kit (SYBR green PCR kit, QIAGEN, Germany). The template cDNA was diluted in RNase-free distilled water to 25 ng / μl. RT-PCR kit (SYBR green PCR kit, QIAGEN, Germany) was used for gene expression analysis. A primer capable of analyzing the expression of each gene was synthesized in Daejeon, Korea. RNase-free distilled water was added to final volume of 20 μl, followed by 15 sec at 94 ° C, 30 sec at 58 ° C, 72 ° C for 30 sec, For 30 seconds and at 65 ° C for 15 seconds as one cycle. At this time, Ct (threshold cycle), which is observed by monitoring the fluorescence signal for each cycle, was analyzed and mRNA expression was quantitatively analyzed with CFX96 Real time system (Bio-Rad, USA). GAPDH was used as an internal transcription marker. The gene primer sequences used for amplification are shown in Table 6 below, and the results are shown in FIG.

gene The forward and reverse primer sequences (53) AdipoR2 Forward GGA CTC CAG AGC CAG ATA TAC GG (SEQ ID NO: 1) Reverse GCC CAT AAA CCC TTC ATC TTC CTG (SEQ ID NO: 2) PPAR Forward GCT GGA GGG TTC GTG GAG TC (SEQ ID NO: 3) Reverse CGG TGA GAT ACG CCC AAA TGC (SEQ ID NO: 4) ACSL Forward CGC ACC CTT CCA ACC AAC AC (SEQ ID NO: 5) Reverse TCG TCG TAG TAG TAC ACC AAG AGC (SEQ ID NO: 6) CPT1 Forward ATC TGG ATG GCT ATG GTC AAG GTC (SEQ ID NO: 7) Reverse GTG CTG TCA TGC GTT GGA AGT C (SEQ ID NO: 8) CPT2 Forward GCC TGC TGT TGC GTG ACT G (SEQ ID NO: 9) Reverse TGG TGG GTA CGA TGC TGT GC (SEQ ID NO: 10) CIDEA Forward GGC TGA TAG GGC AGT GAT TTA (SEQ ID NO: 11) Reverse CGA AGG TGA CTC TGG CTA TTC (SEQ ID NO: 12) PPAR Forward CTG GCC TCC CTG ATG AAT AAA G (SEQ ID NO: 13) Reverse GGT GGG ACT TTC CTG CTA ATA C (SEQ ID NO: 14) SREBF Forward AAC CTC ATC CGC CAC CTG (SEQ ID NO: 15) Reverse TGG TAG ACA ACA GCC GCA TC (SEQ ID NO: 16) FASN Forward CGC TCC TCG CTT GTC GTC TG (SEQ ID NO: 17) Reverse AGC CTT CCA TCT CCT GTC ATC ATC (SEQ ID NO: 18)

As a result, the expressions of AdipoR2, PPARα, ACSL, CPT1 and CPT2, which are involved in fatty acid oxidation, in the high fat control group were significantly lower than those of the normal group, and the expression of the gene was higher in the high fat control group than in the high fat control group , 88%, 77%, 118% and 42%, respectively.

On the other hand, the expression of CIDEA, PPARγ, SREBF and FASN in the high fat control group was significantly increased compared with the normal group, and the expression of the fat synthesis-related genes in the high fat control group was 72%, 40%, and 37% % And 43%, respectively.

4-2. Measurement of lipid metabolism-related enzymatic activity changes by sialic acid supplementation

The liver tissues of the experimental animals collected in Preparation Example 2-3 were pulverized, and then phosphatidic acid phosphatase (PAP), fatty acid synthase (FAS), glucose-6-phosphate dehydrogenase (glucose-6-phophate dehydrogenase, G6PD), β-oxidation and carnitine acyltransferase (CPT) activity were measured.

Specifically, PAP activity was measured according to the method of Walton and Possmayer (1984). That is, 50 μl of a 0.05 M Tris-HCl (pH 7.0), 1.25 mM Na ₂ -EDTA, and 1.0 mM MgCl ₂ and a no-added reaction solution was dissolved in 0.9% NaCl solution to prepare 1 mM phosphatidate and phosphatidylcholine ) Was added thereto, followed by addition of 0.1 mL of enzyme, followed by reaction at 37 ° C for 15 minutes. Then, 0.1 mL of 1.8MH ₂ SO ₄ was added to stop the reaction. Then, 0.25 mL of each of 1.25% ascorbic acid and 0.32% ammonium molybdate was added to the solution and 0.13% sodium dodecyl sulfate solution , And incubated at 45 ° C for 20 minutes. Then, the absorbance was measured at 820 nm.

FAS activity was measured by supplementing and correcting the method performed by Carl et al. (1975). Specifically, the reaction was carried out at 30 ° C with 185.5 mM potassium phosphate buffer solution (pH 7.0), 165 mM acetyl-CoA, 500 nM malonyl-CoA, 500 nM NADPH, 1 uM β-mercaptoethanol and cytoplasmic fractions. Respectively. The activity of FAS was expressed in nmol of NADPH oxidized for 1 minute per 1 mg of cytosolic protein.

G6PD activity was measured and corrected by the method of Pitkanen et al. (1997). After addition of 240 μM NADP ⁺ , 4 mM glucose-6-phosphate and a cytosolic enzyme source in 47.9 mM Tris-HCl buffer (pH 7.3) containing 2.9 mM MgCl ₂ , NADPH The absorbance change was measured. Enzyme activity was expressed in nmol of NADPH produced per minute for 1 mg of cytosolic protein

β-Oxidation activity was measured by Lazarow method (1981) using absorbance as the degree of reduction of NAD ⁺ to NADH with palmitoyl-CoA as substrate. 100 mM Tris-HCl (pH 8.0), 0.2 mM NAD ⁺ , 1.65 mM DTT, 1.5% BSA (bovine serum albumin, 1.5 g / 100 mL), 2% Triton X- After the initiation of the reaction by adding mitochondrial fractions to 100 mM KCN and 5 mM palmitoyl-CoA reaction solutions, the absorbance changes at 37 ° C and 340 nm for 5 minutes were measured. The β-oxidation activity unit was expressed in nmol of NADH produced per minute of 1 mg of mitochondrial protein.

The activity of CPT was determined by measuring the CoASH produced from palmitoyl-CoA using DTNB (5'5-dithiobis- (2-nitrobenzoic acid)) according to the method of Markwell et al. (1973). A mitochondrial fraction was added to a reaction solution containing 97.6 mM Tris-HCl (pH 8.0), 1.1 mM EDTA, 2.50 mM l- carnitine, 0.12 mM DTNB, 0.075 mM palmitoyl-CoA and 0.09% triton X- The absorbance change was measured at 25 ° C and 412 nm for 2 minutes.

The above-mentioned phosphatidic acid phosphatase (PAP), fatty acid synthase (FAS), glucose 6-phophate dehydrogenase (G6PD), beta- and carnitine acyltransferase (CPT) activities were expressed as the mean value per experimental group. A significant difference test was performed using Duncan's multiple range test (DMRT) using one-way ANOVA. The results are shown in Fig.

As a result, the activities of β-oxidation and carnitine acyltransferase (CPT) in the high fat control group were lower than those of the normal group, and the activity of the enzyme in the high fat- Respectively, by 106% and 47%, respectively.

On the other hand, the activity of phosphatidic acid phosphatase (PAP) and fatty acid synthase (FAS) in the high fat control group was higher than that of the normal group, and the activity of the enzyme in the high fat- And 33% and 61%, respectively, compared with the control group.

Example 5: Measurement of Biochemical Index Change by Sicinic Acid Feeding

The activity of fasting glucose, glucose tolerance, asparate aminotransferase (AST) and alanine aminotransferase (ALT) activity, insulin concentration, leptin concentration , Adiponectin concentration and insulin resistance (HOMA-IR) were measured.

Specifically, fasting glucose was measured using a glucose meter (GlucoDr supersensor, Allmedicus, Korea).

The glucose tolerance was determined by collecting blood from the tail vein after 1, 30, 60 and 120 minutes, respectively, after oral administration of 1 g of glucose solution per 1 kg of body weight (kg) (GlucoDr supersensor, Allmedicus, Korea).

The activity of AST (asparate aminotransferase) and alanine aminotransferase (ALT) was measured using a blood biochemical analyzer (Fuji Dri-Chem 3500, Fujifilm, Tokyo, Japan).

The concentrations of insulin, leptin and adiponectin were measured using a mouse insulin ELISA kit (Morinaga ultra sensitive mouse insulin enzyme-linked immunosorbent assay kit, Miobs, Japan), a mouse leptin ELISA kit (Molebs, And adiponectin ELISA kit (Mouse adiponectin duoset, R & D system, USA).

Insulin resistance (HOMA-IR) was calculated using Equation 2 below using fasting glucose and fasting insulin levels.

[Experimental Equation 2]

Insulin resistance (HOMA-IR) = {fasting blood glucose (mmol / L) x fasting insulin (μIU / mL)} / 22.5

The fasting plasma glucose, glucose tolerance, asparate aminotransferase and alanine aminotransferase activity, insulin concentration, leptin concentration, adiponectin concentration and insulin resistance value (HOMA-IR) were expressed as average values per experimental group, Duncan's multiple range test (DMRT) was performed using the -way ANOVA. The results are shown in Fig.

As a result, fasting blood glucose and insulin content of high fat control group were significantly increased from 8 and 12 weeks, respectively, and insulin resistance was induced. However, in the high fat control group (high fat - Siling group) fed with sicolic acid, the insulin concentration was reduced by 43% compared to the high fat control group at 16 weeks, resulting in a 63% reduction in insulin resistance compared to the high fat control group and a 21% reduction in the glucose tolerance .

The activity of AST and ALT in the high fat control group was significantly increased compared with that of the normal group, the concentration of adiponectin was decreased, and the concentration of leptin was increased. However, the ALT activity was decreased by 40%, the adiponectin concentration was increased by 17% compared to the high fat control group, and the leptin concentration was higher than that of the high fat control group (high fat control group %, Which is similar to that of normal group.

Example 6: Measurement of anti-inflammatory activity of serum and liver tissue by feeding sialic acid

6-1. Measurement of Serum Inflammatory Cytokine Activity by Sicalic Acid Supplementation

Twenty microliters of the serum of the experimental animals collected in Preparation Example 2-2 was diluted with TNF-α (Bio-Rad, USA) and Luminex 200 Labmap system (Bio-Rad, USA) (Bio-plex manager software version 5.0 (Bio-Rad)) was used to measure the content of tumor necrosis factor-alpha, IFN-y and interferon-γ and monocyte chemoattractant protein- Respectively. The TNF-α, INF-γ and MCP-1 contents were expressed as the mean value per experimental group. A significant difference test was performed using Duncan's multiple range test (DMRT) using one-way ANOVA. The results are shown in Fig.

As a result, serum TNF-α, INF-γ and MCP-1 levels were significantly increased in the high fat control group compared to the normal control group. However, the contents of TNF-α, INF-γ and MCP-1 were reduced by 54%, 57% and 40%, respectively, in the high fat control group (high fat-seing acid group) fed with sialic acid compared to the high fat control group.

6-2. Measurement of Inflammatory Gene Expression by Sicin-acid Feeding

The expression of inflammation-related genes in liver tissues of the experimental animals collected in Preparation Example 2-3 was measured using the same method as in Example 4-1. The gene primer sequences used for amplification are shown in Table 7 below. The expression of the inflammation-related genes was expressed as a mean value per experimental group. A significant difference test was performed using Duncan's multiple range test (DMRT) using one-way ANOVA. The results are shown in Fig.

gene The forward and reverse primer sequences (53) TLR4 Forward TAT TCA GAG CCG TTG GTG TAT C (SEQ ID NO: 19) Reverse CCA GGT GAG CTG TAG CAT TTA (SEQ ID NO: 20) MYD88 Forward GTA TCC TGC GGT TCA TCA CTA T (SEQ ID NO: 21) Reverse GAA CTC TTC CAC TCA GCT ATC C (SEQ ID NO: 22) NF-B Forward GAA GTG AGA GAG TGA GCG AGA GAG (SEQ ID NO: 23) Reverse CGG GTG GCG AAA CCT CCT C (SEQ ID NO: 24) TNF- Forward AAA GAC ACC ATG AGC ACA GAA AGC (SEQ ID NO: 25) Reverse GCC ACA AGC AGG AAT GAG AAG AG (SEQ ID NO: 26) IL-6 Forward GAG GAT ACC ACT CCC AAC AGA CC (SEQ ID NO: 27) Reverse AAG TGC ATC ATC GTT GTT CAT ACA (SEQ ID NO: 28)

As a result, the expression of inflammatory genes was significantly increased in the high fat control group compared to the normal control group. Expression of inflammatory genes TLR4, MYD88, NF-κB, TNF-α and IL-6 were 58%, 71% and 65%, respectively, in the high fat control group (high fat- , 76% and 69%, respectively.

Example 7: Antioxidant activity measurement by salicylic acid

7-1. Measurement of Antioxidant Metabolism Enzymatic Activity Changes by Sicin Acid

The liver tissues of the experimental animals obtained in Preparation Example 2-3 were pulverized, and then CYP2E1 (cytochrome P450 2E1), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) ) Activity and H ₂ O ₂ (hydroperoxide) and GSH (glutathione) contents were measured.

Specifically, CYP2E1 activity was measured according to the method of Dicker et al. (1990). Specifically, 100 μl of enzyme source, 22.5 μl of 20 mM NADPH, 46 μl of 100 mM aniline and distilled water were added to 45 μl of 1 M potassium phosphate buffer (pH 7.4), and the total volume was adjusted to 450 μl. After the reaction, 90 40 of 40% TCA was added and placed in a cold place for 10 minutes. After centrifugation at 12,000 rpm for 10 minutes at 4 ° C, 360 μl of the supernatant was transferred to a new test tube, and 240 μl of 10% NA ₂ CO _{3 and} 360 μl of 2% phenol were added and left at room temperature for 45 minutes. Respectively. Then, the amount of 4-hydroxyaniline produced was measured and CYP2E1 activity was calculated using a standard calibration curve. The activity of the enzyme was expressed by the amount of 4-hydroxyaniline produced by 1 mg of protein per minute in terms of umol.

SOD activity was modified by Marklund and Marklund (1974) method, and the degree of inhibition of SOD by autoxidation of pyrogallol in alkaline conditions was measured. Specifically, 0.1 mL of enzyme source and 0.1 mL of 7.2 mM pyrogallol solution were mixed well in 1.5 mL of 50 mM Tris-HCl buffer (pH 8.5) containing 10 mM EDTA and reacted at 25 ° C. for 10 minutes. Then, 50 μL of 1N HCl was added The reaction was terminated and the absorbance was measured at 420 nm. SOD activity units were expressed as a unit of 1 mg protein required for 50% inhibition of autoxidation of a 7.2 mM pyrogallol solution which was reacted for 10 minutes without adding an enzyme source.

CAT activity was measured by correcting and complementing the method of Aebi (1988). That is, 2.89 mL of 50 mM potassium phosphate (pH 8.5) and 10 μL of an enzyme source were mixed and reacted at 25 ° C. for 5 minutes. Then, 0.1 mL of 0.3 MH ₂ O ₂ solution was added to measure the absorbance before reaction at 240 nm Then, after further reaction at 25 ° C for 5 minutes, the absorbance was measured at 240 nm to determine the change in H ₂ O ₂ absorbance. The CAT activity unit showed the amount of H ₂ O ₂ lost by 1 mg protein for 1 minute.

The GSH-Px activity was measured by a coupled enzyme procedure using H ₂ O ₂ as a substrate, corrected and supplemented by the method of Paglia and Valentine (1967). That is, reduced glutathione (GSH) reacts with H ₂ O ₂ by GSH-Px to become oxidized glutathione (GSSG), which is again reduced by glutathione reductase and NADPH. At this time, the degree of decrease of absorbance of NADPH at 340 nm was measured to calculate the activity of GSH-Px. Specifically, 0.1 mL of a 30 mM reduced form GSH solution, 0.1 mL of a 6 mM NADPH solution, and 0.1 mL of 25 μM H ₂ O ₂ were sequentially added to 2.6 mL of a 0.1M Tris-HCl buffer solution (pH 7.2), followed by further reaction at 25 ° C for 5 minutes The absorbance was measured after the reaction at 340 nm. The GSH-Px activity unit showed about 1 minute of NADPH oxidation per 1 mg of protein.

The activity of GST is determined by the method of Habig et al. (1974), which results from the reaction of 1-chloro-2,4-dinitrobenzene with reduced glutathione and enzyme source The absorbance of the thioether was measured at 340 nm. The activity of GST was calculated using the extinction coefficient (9.5 M ^-1 cm ^-1 ), and the content of thioether produced in 1 minute of protein was expressed in nmol.

H ₂ O ₂ activity was measured according to the method of Wolff et al. (1994). Hydrogen peroxide (H ₂ O ₂ ) in vivo oxidizes ferrous (Fe ²⁺ ) ions to ferric (Fe ³⁺ ) ions. It uses xylenol orange to oxidize ferric The degree of H ₂ O ₂ production was measured by increasing the absorbance. To 50 μl each of the mitochondrial fractions isolated previously, 950 μl of FOX solution (0.1 M xylenol orange, 0.25 mM ammonium ferrous sulfate, 100 mM sorbitol, 25 mM H ₂ SO ₄ ) was added and reacted at room temperature for 30 minutes The absorbance was measured at 25 ° C and 500 nm and quantified by the content of H ₂ O ₂ standard solution.

GSH activity was measured by supplementing Ellman's method (1959). 0.3 mL of distilled water and 0.5 mL of 4% sulfosalicylic acid were added to 0.2 mL of hepatic cytoplasmic fraction and centrifuged at 4 ° C and 2,500 rpm for 10 minutes. Next, 0.3 mL of the supernatant was taken and reacted with DTNB (5,5'-dithio-bis (2-nitrobenzoic acid)), a coloring agent of sulfhydryl, to quantitate the color at 412 nm. And the amount thereof was calculated by a calibration curve.

The CYP2E1, SOD, CAT, GSH-Px and GST activities, H ₂ O ₂ and GSH contents were expressed as the mean value per experimental group. The significance difference test was performed by Duncan's multiple range test using a one-way ANOVA, DMRT) was performed. The results are shown in Fig.

As a result, CYP2E1 activity and H ₂ O ₂ content in the high fat control group were increased, and CAT, GSH-Px and GST activities were decreased. On the other hand, the activities of CYP2E1 and H ₂ O ₂ in the high fat-seing acid group were decreased by 31% and 29%, respectively, and the CAT, GSH-Px and GST activities were increased by 48%, 40% and 29%, respectively .

7-2. Measurement of lipid metabolism-related protein expression by sialic acid supplementation

(Lysis solution, 50 mM Hepes (pH 7.5), 150 mM NaCl, 1 mM EDTA, 2 mM EGTA, 50 mM NaF, 1% Triton, 1 mM PMSF, 25 μg / mL leupeptin, 2 μg / mL aprotinin) were added and homogenized using a pulverizer. The homogenate was mixed at 4 ° C for 30 minutes, centrifuged at 8,850 × g for 1 hour, and the supernatant was quantitated by Bradford (1976). Then, 50 μg of the protein was electrophoresed on a 10% SDS polyacrylamide gel and then transferred to a nitrocellulose membrane (Whatman, Germany), followed by the addition of 0.1% Tween-20 Tris buffer solution (TBST) for 15 minutes and blocked with 5% BSA solution for 1 hour at room temperature. The primary antibody of AMPK, p-AMPK, ACC, p-ACC (1: 1000, Cell Signaling, USA) and PPARα (1: 200, Santa Cruz, USA) was diluted in 5% And reacted for 24 hours. The next day, the washed nitrocellulose membrane was washed three times for 15 minutes with TBST buffer solution. The washed nitrocellulose membrane was reacted with anti-rabbit IgG (GE Healthcare, USA) secondary antibody for 2 hours at room temperature and then washed three times for 15 minutes with TBST buffer solution After the nitrocellulose membrane was added to ECL solution (Santacruz Biotechnology, USA) for 5 minutes, the cells were scanned and analyzed using LAS 4000 (Fujifilm, USA). The nitrocellulose membrane was washed with TBST buffer solution three times for 15 minutes, and then 700 μl of 2-mercaptoethanol was added to a stripping buffer solution (62.5 mM Tris, 2% SDS, pH 6.7) (1: 200, Sigma, St. Louis, Mo., USA) was measured in the same manner as the other antibodies. Protein expression level was calculated after correction with β-actin.

The lipid metabolism-related protein expression was expressed as the mean value per experimental group, and significant difference test was performed by Duncan's multiple range test (DMRT) using one-way ANOVA. The results are shown in Fig.

As a result, the expression of p-AMPK was increased by about 20% and the expression of p-ACC was increased by about 68% in the high fat-Searing acid group, compared with the high fat control group. In addition, the expression of PPARα, a fatty acid oxidation factor, was increased by about 15% as compared with the high fat control group.

<110> Chosun University Industry-Academic Cooperation Foundation          Chosun Natural Pharmaceutical Material Development Research Center <120> Compositions for preventing or treating obesity or          lipid-related metabolic disease comprising syringic acid, or salt          thereof as an active ingredient <130> PA-14-0150 <160> 28 <170> Kopatentin 2.0 <210> 1 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> AdipoR2 Forward primer <400> 1 ggactccaga gccagatata cgg 23 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> AdipoR2 Reverse primer <400> 2 gcccataaac ccttcatctt cctg 24 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PPAR alpha Forward primer <400> 3 gctggagggt tcgtggagtc 20 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PPAR alpha reverse primer <400> 4 cggtgagata cgcccaaatg c 21 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ACSL Forward primer <400> 5 cgcacccttc caaccaacac 20 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> ACSL Reverse primer <400> 6 tcgtcgtagt agtacaccaa gagc 24 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> CPT1 Forward primer <400> 7 atctggatgg ctatggtcaa ggtc 24 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CPT1 Reverse primer <400> 8 gtgctgtcat gcgttggaag tc 22 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> CPT2 Forward primer <400> 9 gcctgctgtt gcgtgactg 19 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CPT2 Reverse primer <400> 10 tggtgggtac gatgctgtgc 20 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CIDEA Forward primer <400> 11 ggctgatagg gcagtgattt a 21 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CIDEA Reverse primer <400> 12 ggctgatagg gcagtgattt a 21 <210> 13 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PPAR gamma Forward primer <400> 13 ctggcctccc tgatgaataa ag 22 <210> 14 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PPAR gamma reverse primer <400> 14 ggtgggactt tcctgctaat ac 22 <210> 15 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> SREBF Forward primer <400> 15 aacctcatcc gccacctg 18 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SREBF Reverse primer <400> 16 tggtagacaa cagccgcatc 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FASN Forward primer <400> 17 cgctcctcgc ttgtcgtctg 20 <210> 18 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> FASN Reverse primer <400> 18 agccttccat ctcctgtcat catc 24 <210> 19 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> TLR4 Forward primer <400> 19 tattcagagc cgttggtgta tc 22 <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> TLR4 Reverse primer <400> 20 ccaggtgagc tgtagcattt a 21 <210> 21 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> MYD88 Forward primer <400> 21 gtatcctgcg gttcatcact at 22 <210> 22 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> MYD88 Reverse primer <400> 22 gaactcttcc actcagctat cc 22 <210> 23 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> NF-kappaB Forward primer <400> 23 gaagtgagag agtgagcgag agag 24 <210> 24 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> NF-kappaB Reverse primer <400> 24 cgggtggcga aacctcctc 19 <210> 25 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> TNF-alpha Forward primer <400> 25 aaagacacca tgagcacaga aagc 24 <210> 26 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> TNF-alpha Reverse primer <400> 26 gccacaagca ggaatgagaa gag 23 <210> 27 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> IL-6 Forward primer <400> 27 gaggatacca ctcccaacag acc 23 <210> 28 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> IL-6 Reverse primer <400> 28 aagtgcatca tcgttgttca taca 24

Claims (4)

A pharmaceutical composition for preventing or treating obesity comprising syringic acid or a pharmaceutically acceptable salt thereof as an active ingredient.
delete A food composition for preventing or ameliorating obesity comprising syringic acid or a pharmaceutically acceptable salt thereof as an active ingredient. delete

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