KR101368723B1 - Composition and functional food for prevention and treatment of obesity - Google Patents

Abstract

The present invention relates to a composition and a functional food containing the aroma extract as an active ingredient, the aroma extract inhibits excessive lipid production, differentiation of fat cells and fat accumulation, so that various diseases related to obesity and excessive lipid production, such as It is useful for the prevention and treatment of hyperlipidemia or fatty liver.

Description

Composition and functional food for prevention and treatment of obesity

The present invention relates to compositions for the prevention and treatment of diseases caused by excessive lipid formation and accumulation, including obesity.

Obesity is an imbalance between calorie intake and consumption, resulting in excessive accumulation of adipose tissue, which is closely related to diabetes, hyperlipidemia, hypertension, and coronary artery disease. In addition, obesity has been reported to increase the prevalence and mortality of colorectal cancer, prostate cancer, gallbladder cancer, cervical cancer, endometrial cancer, ovarian cancer, breast cancer, gallstones and cholangiopathy, osteoarthritis, menstrual irregularities and hypermenorrhea, psychological disorders. And many other health problems (Garfinkel L. Overweight and cancer. Ann Intern Med, 103: 1034-6, 1985). Therefore, the treatment of obesity is considered to be a very effective means for the treatment and prevention of adult diseases (Lee Heung-kyu, obesity-related diseases, Korean Journal of Nutrition, 23; 5 pp341-346,1990; William BK, Ralph BD, Janet LC. Effect of weight on cardiovascular disease, AM J Clin Nutr, 63; 419S-9S, 1996).

Obese patients have very developed adipose tissue, and the number and size of adipocytes are significantly increased compared to normal people. Increasing the number and size of these fat cells increases the ability to store triglycerides in the event of overnutrition. Therefore, suppressing the differentiation of adipocytes or inhibiting the function of absorbing excessively ingested nutrients and accumulating into fats can provide a beneficial means for treating or preventing obesity.

Fat cells store a lot of fat and act as a cause of obesity, and secrete hormones, growth factors, and cytokines as endocrine cells to cause complications due to obesity. Factors secreted by adipocytes are called adipocytokines.

Leptin, a representative adipocytokine expressed in adipocytes, is an indicator of adipocyte differentiation, and its expression and secretion have an effect of suppressing obesity. Leptin mainly acts on the hypothalamus to reduce the expression of orexigenic peptides, thereby inhibiting the activity of related neuropeptide Y (NPY), and increasing the expression of anorexic peptides to increase the expression of anorexic peptides. -opiomelanocortin) inhibits appetite and weight gain by controlling food intake (Ahima RS, Filer JS, Leptin. Annu Rev Physiol 62: 413-437, 2002; Kolaczynski JW, Ohannesian J, Considine RV. leptin to short-term and prolonged overfeeding in humans.J Clin Endocrinol Metab 81: 4162-4165, 1996). In ob / ob mice, the ob / ob mouse lacks leptin genes, which prevents the expression of leptin protein. As a result, ob / ob mice show increased food intake, increased body weight, increased body fat mass, reduced energy consumption, and reduced immune function. , Impotence, etc. (Jeffery M, Friedman Jeffery L, Halaas: Nature 395: 763-770, 2000). Adipocytokines secreted by adipocytes include TNF-alpha, IL-6, macrophage colony-stimulating factor (MCSF), and plasminogen activator inhibitor-1 (PAI-). 1), angiotensinogen, tissue factor, transforming growth factor (TGF-β), adiponectin, resistin, and the like are known. There are various complications such as increased thrombosis, increased atherosclerosis, increased diabetes, and increased hyperlipidemia.

A representative transcription factor required to increase adipocyte differentiation, energy uptake and lipid production is PPAR gamma, known as Peroxysome proliferator activated receptors (PPARs). It is a type of nuclear hormone receptor superfamily known to be a class of families such as PPAR alpha and PPAR delta, and these PPAR transcription factors form heterodimers with Retinoid X receptor (RXR) alpha to promote the target gene or The peroxisome proliferator response element (PPRE) in the enhancer is recognized and the expression of the target gene is controlled. They increase expression in adipocytes during obesity. In addition, C / EBP alpha is an important transcription factor that acts on the differentiation and size increase of adipocytes. When C / EBP alpha and PPAR gamma increase, expression of adipocyte-specific proteins LPL, aP2, FAS, GPDH and GLUT4, etc. This promotes obesity (Stephens JM, Pekala PH, Transcriptional repression of the GLUT 4 and C / EBP genes in 3T3-L1 adipocytes by tumor necrosis factor-α. J Biol Chem 266: 21839-21845,1991). In addition, FATP-1, FAS, CTP-1, ACS-1, etc. play a role of accumulating fat in adipocytes through the absorption and metabolism of lipids. HMG coenzyme A reductase is also an important enzyme for biosynthesis of cholesterol. Therefore, inhibiting the expression of such factors inhibits the accumulation of fatty acids and cholesterol in adipose tissue, thereby exhibiting an obesity treatment and prevention effect.

Aquilaria agallocha Roxb. Is an evergreen tree of the Aquilaria genus Thymelaeaceae in Southeast Asia, including India, Vietnam, Laos, and China. It protects against injuries and invasion of bacteria and fungi. Produces a resin component, and the tree where the resin component is deposited is aroma. Aroma has long been used as a medicinal herb or fragrance. In oriental medicine, it has been used for soothing, dry stomach, aeration, indigestion, anorexia, vomiting, bronchial asthma, premature ejaculation and lack of energy. As a pharmacological activity of acupuncture, it inhibits the central nervous system (Okugawa H., Ueda R., Matsumoto K., Kawanishi K., Kato A .: Effects of agarwood extracts on the central nervous system in mice, Planta Medica, 59, p32, 1993) and lowering action (Kakino M, Tazawa S, Maruyama H, Truruma K, Araki Y, Shimazawa M, Hara H, Laxative effects of agarwood on low-fiber diet-induced costipation in rats, BMC Compementary and Alternative Medicine, 10, Art. No. 68, 2010), and other antimicrobial effects (Dash M, Patra JK, Panda PP, Phytochemical and antimicrobial screening of extracts of Aquilaria agallocha Roxb, African J. of Biotechnology, 7 (20), pp 3531-3534, 2008), anticancer effect (Gunasekera SP, Kinghorn AD, Cordell GA, Farnsworth NR, Plant anticancer agents.XIX.Constituents of Aquilaria Malaccensis.J. of Natural Products, 44 (5), pp569-572, 1981 ), Anti-inflammatory effects (Antinociceptive and anti-inflammatory activiteies of Aquilaria sinensis (Lour.) Gilg.Leave extract, Zhou MH, Wang H G, Suolangjiba, Kou JP, Yu BY, J. of Ethnopharmacology, 117 (2), pp 345-350), Effect of the aqueous extract of Aquilaria agalloch stems on the immediate hypersensitivity reactions, J. of Ethnopharmacology, 58 (1), pp31-38, Kim YC, Lee EH, Lee YM, Kim HK, Song BK, Lee EJ, Kim HM). Aromas are mixed with other herbal medicines to make herbal tonics or herbal beverages (Korean Patent No. 071336), blood circulation promoting health foods (Korean Patent No. 076306), atopy prevention (Korean Patent No. 0894439) or collagen synthesis It has been developed as a cosmetic (Korean Patent No. 0309071) that has an effect of promoting and the like, a herbal drink for relieving heart disease and depression (Korean Patent No. 0913880), and an insecticide (Korean Patent No. 0486819). However, no research has been reported or reported on the anti-obesity activity of aromas.

Currently, many studies are being conducted to develop obesity treatments, and obesity is suppressed by suppressing appetite (e.g., reductyl of sibutramine) or inhibiting fat absorption by inhibiting pancreatic lipase (e.g., oral-statin). Therapeutic agents are commercially available and show excellent effects. However, despite these excellent effects, some patients have reported side effects such as stroke, heart attack, gastrointestinal disorders, hypersensitivity, biliary secretion, and fat-soluble vitamin absorption (Peter C. et al., 2001, Br. J.). Clin. Pharmacol. 51: 135-141). Therefore, efforts have recently been made to develop anti-obesity materials using foods or natural products that have no side effects. For example, dietary fibers such as hydroxycitric acid (HCA), chicory, inulin, and the like are involved in appetite suppression, and chitosan and flavonoids are substances that inhibit digestion and absorption of fat. Other substances that induce heat generation and inhibit fat accumulation include capsaicin of red pepper, catechin of green tea, L-cartitine, conjugated linoleic acid (CLA), milk calcium and related proteins that regulate lipid metabolism. And the like have been reported. Herbal medicines such as fruit, yangha, deficiency, green tea, pine needles, hwahwa, cheongung, and filthy milk have been reported to be effective for weight control (Kim MH, 2004, Korean J. Heath Psychol. 9: 493-509, Reddy P. et al., 1998, Formulary 33: 943-959, Burns AA et al., 2002, Eur. J. Clin. Nutr. 56: 368-377, Delzenne NM et al., 2005, Br. J. Nutr 1: 157-161, Zacour AC et al, 1992, J. Nutr. Sci. Vitamilol. 38: 609-613, Griffiths DW et al., 1986, Adv.Exp. Med. Biol. 1999: 509-516) . Therefore, consumers are more interested in obesity treatments based on natural products with excellent medicinal effects and fewer side effects, and in order to meet these demands, development of safe and effective natural medicines is desired.

The inventors have surprisingly found that aromas can be useful as medicines or health functional foods for the treatment and prevention of diseases caused by excessive lipid formation such as obesity by inhibiting adipocyte differentiation, excessive lipid formation and fat accumulation. Completed.

Republic of Korea Registered Patent No. 071336 (Published 20 December 2006) Republic of Korea Patent Registration No. 0376306 (March 17, 2003) Republic of Korea Patent Registration No. 0894439 (Announcement dated April 22, 2009) Republic of Korea Patent No.0309071 (Published April 6, 2000) Republic of Korea Registered Patent No. 0927880 (December 15, 2009 registration notification) Republic of Korea Patent No. 0486819 (published 23 November 2002)

Garfinkel L. Overweight and cancer. Ann Intern Med, 103: 1034-6, 1985 Lee Heung-kyu. Obesity-related Diseases, Korean Journal of Nutrition, 23; 5 pp341-346,1990 William BK, Ralph BD, Janet LC. Effect of weight on cardiovascular disease, AM J Clin Nutr, 63; 419S-9S, 1996 Ahima RS, Filer JS, Leptin. Annu Rev Physiol 62: 413-437, 2002 Kolaczynski JW, Ohannesian J, Considine RV. Response to leptin to short-term and prolonged overfeeding in humans. J Clin Endocrinol Metab 81: 4162-4165, 1996 Jeffery M, Friedman Jeffery L, Halaas: Nature 395: 763-770, 2000 Stephens JM, Pekala PH, Transcriptional repression of the GLUT 4 and C / EBP genes in 3T3-L1 adipocytes by tumor necrosis factor-α, J Biol Chem 266: 21839-21845, 1991 Okugawa H., Ueda R., Matsumoto K., Kawanishi K., Kato A .: Effects of agarwood extracts on the central nervous system in mice, Planta Medica, 59, p32, 1993 Kakino M, Tazawa S, Maruyama H, Truruma K, Araki Y, Shimazawa M, Hara H, Laxative effects of agarwood on low-fiber diet-induced costipation in rats, BMC Compementary and Alternative Medicine, 10, Art. No. 68, 2010 Dash M, Patra JK, Panda PP, Phytochemical and antimicrobial screening of extracts of Aquilaria agallocha Roxb, African J. of Biotechnology, 7 (20), pp 3531-3534, 2008 Gunasekera S.P., Kinghorn A.D., Cordell G.A., Farnsworth N.R., Plant anticancer agents. XIX. Constituents of Aquilaria Malaccensis. J. of Natural Products, 44 (5), pp 569-572, 1981 Zhou MH, Wang HG, Suolangjiba, Kou JP, Yu BY, Antinociceptive and anti-inflammatory activiteies of Aquilaria sinensis (Lour.) Gilg. Leaves extract, J. of Ethnopharmacology, 117 (2), pp 345-350 Kim YC, Lee EH, Lee YM, Kim HK, Song BK, Lee EJ, Kim HM, Effect of the aqueous extract of Aquilaria agalloch stems on the immediate hypersensitivity reactions, J. of Ethnopharmacology, 58 (1), pp31-38 Peter C. et al., 2001, Br. J. Clin. Pharmacol. 51: 135-141 Kim M. H., 2004, Korean J. Heath Psychol. 9: 493-509, Reddy P. et al., 1998, Formulary 33: 943-959 Burns A. A. et al., 2002, Eur. J. Clin. Nutr. 56: 368-377 Delzenne N. M. et al., 2005, Br. J. Nutr. 1: 157-161 Zacour A. C. et al, 1992, J. Nutr. Sci. Vitamilol. 38: 609-613 Griffiths D. W. et al., 1986, Adv. Exp. Med. Biol. 1999: 509-516

An object of the present invention is to provide a composition for the treatment and prevention of obesity, and a functional food, which is a safe and effective natural medicine.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention or treatment of diseases selected from the group consisting of obesity, hyperlipidemia and fatty liver containing aroma or aroma extract as an active ingredient.

In the present invention chimhyang may be used originated from Aquila Liao (Aquilaria) in plants, for example, Liao Aquila or cassia (A. khasiana), Aquila Ria Bahia ku Lina (A. apiculina), carbonyl (A to Aquila Ria Bulletin . blillonil), Aquila Ria bar neon system (A. baneonsis), Aquila Ria beuraki hits (A. brachyantha), Aquila Ria keonming the ANA (A. cunmingiana), Aquila RIA RIA pillar (A. filaria), Aquila Ria Grandi Flora (A. grandiflora), Aquila Ria Hill rata (A. hilata), Aquila Ria micro kappa (A. microcapa), Aquila Ria roast rata (A. rostrata), by RIA Aquila Agar car (A. agallocha), Aquila Ria A. malaccensis , A. sinensis , or Stems or roots, such as A. crassna , may be used, more preferably stems or roots on which resin is deposited.

Aroma extracts according to the present invention can be prepared by extracting aromas by cold water extraction, hot water extraction, organic solvent extraction or distillation according to a conventional manufacturing method, and then using the obtained extract as it is, or by concentrating under reduced pressure or lyophilization. Can be. For example, the aroma is obtained by extracting with a solvent selected from the group consisting of water, lower alcohols and mixed solutions thereof. Alternatively, the fragrance oil fraction may be prepared by distilling the fragrance, cooling the distillate, and taking only the oil layer. The fragrance extract of the present invention not only means the material itself obtained by extracting water or a specific solvent from the incense, but also sometimes the material obtained by extracting is fermented through a strain, or further with an organic solvent such as ether. By fractional extraction or fractionation through a column (column) it may mean an extracted processed material, such as processed. Specifically, it may be prepared as follows.

First, about 1 to 50 times the amount of water, ethanol, methanol, etc., C ₁ to C ₄ lower alcohols, or a mixed solvent thereof, based on the weight of the fragrances selected and trimmed to an appropriate size, for example, 5 ° C. to 100 ° C. For about 1 to 100 hours, extracts obtained by using extraction methods such as stirring extraction, boiling water extraction, cold needle extraction, reflux extraction or ultrasonic extraction can be filtered, concentrated or dried to obtain aroma extract. If necessary, the extract may be further extracted with water or an organic solvent such as lower alcohol, ether, or the like.

Or finely pulverize the incense or incense wood, soaked in water for 1 to 15 days, put into a distillation, and then put in a distiller, the steam from the slowly heated together with water is cooled through a cooler to separate the distilled oil and water to obtain a fragrance oil. Can be. The aroma oil obtained in this way contains mainly volatile substances of aroma, and its efficacy is similar to the fraction and components extracted from the total extract of aroma with ether or hexane.

Fragrance extract of the present invention, as shown in the experimental example below, genes of factors involved in the absorption and metabolism of lipids and differentiation of fat cells, such as PPAR gamma, C / EBP, FATP-1, SREBP-1, CTP-1 Reduced expression significantly inhibits excessive lipid formation, differentiation of fat cells and fat accumulation.

On the other hand, pharmaceutical compositions or nutraceuticals containing the fragrance extract according to the present invention as an active ingredient can be prepared through a conventional formulation method. In this case, in addition to the aroma extract, various auxiliary herbal medicines, vitamins, minerals, dietary fiber, and other medicinal-food supplements (eg, excipients, enhancers, coatings, etc.) may be contained.

The pharmaceutical composition according to the present invention can be usefully used for the prevention and treatment of various diseases related to excessive lipid formation, such as obesity, hyperlipidemia or fatty liver, by containing aroma extracts and the like. The pharmaceutical composition according to the present invention may include a fragrance extract as an active ingredient, and a pharmaceutically acceptable carrier may be added according to a pharmaceutical preparation method known in the art. Examples of carriers usable in the present invention include lactose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, poly Vinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like, but are not limited thereto. In addition, the lipase inhibitor may further include fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers or preservatives. The pharmaceutical compositions according to the present invention may be used in the form of oral dosage forms, external preparations, suppositories, and sterile injectable solutions, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, respectively, according to conventional methods. Can be. The pharmaceutical composition containing the aroma extract of the present invention can be used clinically for the prevention and treatment of obesity, control of triglycerides in blood, lowering cholesterol levels, improving blood pressure, treating hyperlipidemia and diabetes. Patient dosage, frequency and time of administration of the aroma extract according to the present invention can be selectively added and subtracted and adjusted according to the conditions such as the sex, age, condition, weight, environment of the patient, but generally between meals three times a day It can be administered in one to several times to be administered in an amount of 0.1 mg to 1000 mg.

The functional food according to the present invention can exhibit an improvement effect on obesity inhibition, hyperlipidemia, fatty liver, etc. by containing aroma extracts. In the present invention, the food is a general concept including foodstuffs, food additives, food ingredients, etc., as well as those directly ingestible. The functional food according to the present invention may be used by directly adding the above-mentioned aroma extract alone, but may also exhibit synergism by adding a known substance or a new substance used for preventing and treating obesity, hyperlipidemia and the like. In addition, general additives such as food coloring, food flavoring, herbal medicine, fruit, oligosaccharide, chitosan, citric acid, etc. may be used to give a good taste and aroma.

 Functional foods containing the aroma extract of the present invention include confectionery such as bread, rice cakes, dried fruits, candy, chocolates, chewing gum, and nuts; Ice cream products such as ice creams, ice creams, and ice cream powders; Dairy products such as milks, low fat milks, lactose digested milk, processed milk, goat milk, fermented milk, butter milk, concentrated milk, milk cream, butter oil, natural cheese, processed cheese, milk powder, whey; Meat products such as meat products, processed products, and hamburgers; Fish meat products such as fish meat products such as fish paste, ham, sausage and bacon; Noodles such as ramen noodles, dried noodles, raw noodles, fried noodles, dehydrated noodles, refined noodles, frozen noodles and pasta; Beverages such as fruit beverages, vegetable beverages, carbonated beverages, lactic acid bacteria beverages such as soy milk and yogurt, mixed beverages; Seasoning foods such as soy sauce, miso, red pepper paste, chunjang, cheongukjang, mixed soy sauce, vinegar, sauce, tomato ketchup, curry, and dressing; Margarine, shortening; And pizza, but are not limited thereto.

Hereinafter, the present invention will be described in more detail based on Examples and Experimental Examples. However, these Examples and Experimental Examples are only intended to help the understanding of the present invention, the scope of the present invention is not limited by these Examples.

Fragrance extract of the present invention inhibits excessive lipid production, differentiation of fat cells and fat accumulation, can be usefully used for the prevention and treatment of various diseases related to obesity and excessive lipid production, such as hyperlipidemia or fatty liver.

1 shows aromatized methanol extract (ACR-M), butanol extract (ACR-B), water extract (ACR-W), ether extract (ACR-E), and aroma oil (ACR-O) on adipocytes before differentiation. Image results of fat particles accumulated in adipocytes after addition and differentiation (control: no aroma extract).
Figure 2 shows the effect of the aroma extract on the survival of 3T3-L1 adipocytes.
Figure 3 shows the effect of aroma extracts on the gene expression of leptin (ND: 3T3-L1 adipocytes before differentiation, D: 3T3-L1 adipocytes after differentiation without aroma extracts, ACR-E: aroma extracts 3T3-L1 adipocytes after added differentiation).
Figure 4 shows the effect of aroma extracts on the gene expression of PPAR gamma (ND: 3T3-L1 adipocytes before differentiation, D: 3T3-L1 adipocytes after differentiation without aroma extracts, ACR-E: aroma extracts 3T3-L1 adipocytes after this added differentiation).
Figure 5 shows the effect of aroma extracts on LPL gene expression (ND: 3T3-L1 adipocytes before differentiation, D: 3T3-L1 adipocytes after differentiation without aroma extracts, ACR-E: aroma extracts added 3T3-L1 adipocytes after differentiation).
Figure 6 shows the effect of aroma extracts on C / EBP alpha gene expression (ND: 3T3-L1 adipocytes before differentiation, D: 3T3-L1 adipocytes after differentiation without aroma extracts, ACR-E: aroma 3T3-L1 adipocytes after differentiation with addition of extract).
Figure 7 shows the effect of the aroma extract on the gene expression of ACS-1 (ND: 3T3-L1 adipocytes before differentiation, D: 3T3-L1 adipocytes after differentiation without aroma extracts, ACR-E: aroma 3T3-L1 adipocytes after differentiation with addition of extract).
Figure 8 shows the effect of aroma extract on FAS gene expression (ND: 3T3-L1 adipocytes before differentiation, D: 3T3-L1 adipocytes after differentiation without aroma extracts, ACR-E: aroma extracts added 3T3-L1 adipocytes after differentiation).
Figure 9 shows the effect of aroma extract on FATP-1 gene expression (ND: 3T3-L1 adipocytes before differentiation, D: 3T3-L1 adipocytes after differentiation without aroma extract, ACR-E: aroma extract 3T3-L1 adipocytes after this added differentiation).
Figure 10 shows the effect of aroma extracts on aP-2 gene expression (ND: 3T3-L1 adipocytes before differentiation, D: 3T3-L1 adipocytes after differentiation without aroma extracts, ACR-E: aroma extracts 3T3-L1 adipocytes after this added differentiation).
111 shows the effect of the aroma extract on the gene expression of SREBP-1 (ND: 3T3-L1 adipocytes before differentiation, D: 3T3-L1 adipocytes after differentiation without aroma extracts, ACR-E: aroma 3T3-L1 adipocytes after differentiation with addition of extract).
Figure 12 shows the effect of aroma extract on the gene expression of CTP-1 enzyme (ND: 3T3-L1 adipocytes before differentiation, D: 3T3-L1 adipocytes after differentiation without aroma extract, ACR-E: 3T3-L1 adipocytes after differentiation with added aroma extract)

The full name of the abbreviation used herein is as follows.

PPARγ2: Peroxysome Proliferative Active Receptor Subtype γ2

        (peroxisome proliferator activated receptor subtype γ2)

SREBP1: Sterol-regulatory Element-binding Protein

        (sterol-regulatory element-binding protein) 1

C / EBP-α: CCAAT / Enhancer Binding Protein-alpha

          (CCAAT / enhancer binding protein-alpha)

aP2: amplified mouse fatty acid binding protein

     (amplifying mouse fatty acid binding protein; also called FABP) 2

LPL: Lipoprotein lipase

FATP-1: FA transport protein 1

FAS: Fatty acid synthase

CTP-1: Carnitine palmitoyltransferase 1

ACS-1: acyl-CoA synthetase-1

GPDH: Glyceraldehyde 3-phosphate dehydrogenase

GLUT4: Glucosr transporter type 4

Example 1 . Aroma  Preparation of extract

After 400 g of needles were refluxed three times for 5 hours with 5 L of 70% methanol, the extract was concentrated to give a methanol extract (ACR-M). Water and ether were added to the methanol extract for fractional extraction to obtain an ether extract. Butanol was added to the remaining water layer for fractional extraction to obtain butanol extract and water extract. The extracts were concentrated under reduced pressure, respectively, to prepare an ether extract (ACR-E), a water extract (ACR-W), and a butanol extract (ACR-B).

Example 2 . Aroma  Preparation of extract

After crushing 100 g of fine fragrance, immersed in water for 3 days, and then put in a steam distillation apparatus to steam distillation to prepare a fragrance oil (ACR-O).

The reagents isobutyl methylxanthine, dexamethasone, insulin and MTT used in the following experiment were purchased from Sigma (St. Louis, MO). The RNAiso Plus reagents used for RNA extraction, the Primescript TM1ststrandc DNA Synthesis kit and the SYBR Premix Ex TaqTM realtime PCR kit for cDNA synthesis and real-time polymerase chain reaction are available from TAKARA BIO Inc. It was purchased from (Otsu, Shiga, JAPAN). The following experiments were performed three times and all results are expressed as mean + standard error (mean ± SEM (n = 3)). Student's -test was used to confirm the statistical significance of each experiment and was defined as significant if the probability was less than 0.05.

Experimental Example  1. Cell Culture and Adipocyte Differentiation

Adipocytes (purchased from the American Type Culture Collection) before 3T3-L1 differentiation were cultured in DMEM medium containing 10% eluted serum (GIBCO BRL) (WelGENE, Daegu, KOREA). The temperature of the incubator was maintained at 37 ° C. and 5% carbon dioxide concentration was maintained. Two days later (day 0 of differentiation), cells were packed in culture plates and exchanged with DMEM differentiation medium containing MDI (1 μg / mL of isobutyl methylxanthine, 1 μM of dexamethasone, 1 μg / mL of insulin). Two days later (differentiation 2 days), MDI medium was exchanged again with DMEM medium containing only insulin. Two days later (day 4 of differentiation), insulin-DMEM medium was replaced with DMEM medium containing 10% fetal calf serum (FBS, Lonza, Walkersville, MD USA) and cultured for 5 more days (day 9 of differentiation). Methanol, butanol, water, ether extract and aroma oil of the aroma prepared in Example 1 were dissolved in DMSO or water, respectively, so that the final treatment concentration was 10 μg / mL, and was added to the medium during the differentiation period.

Image of fat particles formed in differentiated cells after aliquotation of 2.5 × 10 ⁵ adipocytes 3T3-L1 into 6-well plates prior to differentiation and differentiation for 9 days with or without the above-mentioned aroma extract. Was obtained using an inverted microscope OLYMPUS, IX 51 (Olympus Co., Tokyo, JAPAN), 100x magnification. The image obtained by adding the ether extract (ACR-E) is shown in FIG. 1. As shown in FIG. 1, in the case of the control group to which no aroma extract was added, a large amount of fat particles were formed, but when the aroma extract was added, the number of fat particles formed in the cells was significantly reduced. In particular, it was found that the ether extract (ACR-E) and aroma oil (ACR-O) of aromas effectively inhibit adipocyte differentiation. The study was then performed using the most effective ether extract (ACR-E) and the results are shown in FIGS. 2-12.

Experimental Example  2. Aroma  Effect of Extracts on Cell Survival ( MTT  Search method)

The effect of aroma extracts on the survival of 3T3-L1 was examined using the color reaction (MTT) using tetrazolium. Dispense 0.5 × 10 ⁵ 3T3-L1 cells into 24-well plates and add acupuncture extract (ACR-E) at 0.01, 0.1, 1, 10 μg / mL or for 9 days without Differentiated. Differentiated 3T3-L1 cells were washed twice with saline, and 100 ml of MTT solution was added and then incubated for 1 hour in a 37 ° C, 5% carbon dioxide incubator. After removing the MTT solution, the solution was washed once with brine, 100 ml of DMSO was added to extract the reaction solution, and the absorbance was measured at 570 nm using a microplate reader (GENios A5082, TECAN, Maennedort, Switzerland).

As shown in FIG. 2, the aroma extract had little effect on the survival of fat cells. There was no significant decrease in cell viability even at a concentration of 10 μg / mL which effectively inhibited intracellular fat formation. From this, it was confirmed that the aroma extract is an effective and safe substance that inhibits differentiation into fat cells without affecting cell survival.

Experimental Example 3. RNA extraction and real-time polymerase chain reaction (Quantitative realtime PCR )

Dispense 3T3-L1 cells into 6-well plates into 2.5 × 10 ⁵ cells prior to differentiation, and differentiate for 9 days with or without 10 μg / mL of salivary extract (ACR-E) and total RNA. Was extracted. Specifically, the differentiated cells were washed twice with saline, and 4.00 µl of RNAiso reagent was added to lyse the cells. The obtained cell lysate was centrifuged to separate the RNA layer, chloroform was added to remove the protein, and only RNA was precipitated with alcohol. Total RNA obtained was NonoDrop  Concentration and purity were measured using an ND-1000 (NanoDrop Technologies, Inc., Rockland DE) instrument. A total RNA of 1 mg or less was used as a template to make complementary cDNA. cDNA was synthesized using PrimeScript ^™ 1st strand cDNAsynthsis kit (Takarasa, Japan). Briefly, 5 mM random 6-primer, each 1 mM dNTP solution, and template RNA were reacted at 65 ° C. for 5 minutes, transferred to ice, and then added to the reaction solution with 1 U / ml RNase inhibitor, 10 U / ml PrimeCscript ^TM. Reverse transcriptase, PrimeScript ^™ reverse transcriptase buffer was added. It was reacted at 30 ° C. for 10 minutes and again at 42 ° C. for 60 minutes. To compare the expression patterns of genes that change during the differentiation of adipocytes, real-time polymerase chain reaction was performed with the synthesized cDNA. SYBR premix Ex Taq ^™ kit (Takara, Japan) was used for the reaction, and the results were analyzed by TAKARA Dice Real Time System. At the beginning of the reaction, the cDNA was denatured at 95 ° C. for 10 seconds, and amplified by 95 cycles for 5 seconds at 95 ° C., 30 seconds at 55-60 ° C., and 40 cycles. The expression levels of the amplified genes were compared and analyzed with the messenger RNA levels of the β-actin gene, followed by electrophoresis on a 1.5% agarose gel to image band density.

Primers of the genes involved in the real-time polymerase chain reaction of genes involved in the process of differentiation into adipocytes and genes expected to be changed after differentiation are shown in Table 1 below.

MRNA levels of the genes were analyzed and shown in FIGS. 3 to 12, respectively.

Figure 3 shows the gene expression level of leptin, adipocytokine specifically found in adipocytes. Leptin is a marker of adipocyte differentiation and, as evidenced by several experiments, increased gene expression of intracellular leptin as it differentiated into adipocytes (D). On the other hand, in the case of the cells to which the aroma extract was added (ACR-E), the expression of leptin was significantly suppressed to almost pre-differentiation level (ND). As a result, it was confirmed that the aroma extract effectively inhibits the differentiation into adipocytes.

Figure 4 shows the observed gene expression changes of PPAR gamma, a representative transcription factor involved in energy metabolism and lipid synthesis in differentiated adipocytes. Cells treated only with differentiation medium (D) had completed differentiation into adipocytes and expressed more PPAR gamma than pre-differentiated cells (ND), but in the cells added with aroma extract (ACR-E) to adipocytes It was confirmed that the expression of PPAR gamma, a representative factor, was markedly suppressed by differentiation of about 2.5-fold inhibition.

5 shows a comparison of gene expression levels of LPL. LPL is an enzyme that breaks down fat in the blood and accumulates triglycerides in adipocytes. As expected, the expression level in adipocytes increased (D), and when treated with aroma extract (ACR-E), pre-differentiation levels ( ND) significantly inhibited the gene expression of LPL.

FIG. 6 illustrates the expression level of C / EBP alpha, a transcriptional regulator that regulates transcription of various genes regulated at the early stage of differentiation. Significantly decreased (ACR-E). On the other hand, the cells treated with differentiation medium were able to confirm that the differentiation progressed because C / EBP gene was expressed at a higher level than before differentiation (D). In other words, the aroma extracts were found to reduce C / EBP alpha at the level of transcription that regulates the transcription of genes appearing in the early stages of adipocyte differentiation.

Figure 7 shows the gene expression changes of ACS-1 by the aroma extract. ACS-1 is an enzyme that converts long-chain fatty acids into CoA derivatives before they enter the private body for beta oxidation. In obese animals or patients, ACS-1 is highly saturated with expression in fat cells. In the present results, the gene expression of ACS-1 was significantly increased after differentiation into adipocytes (D), but it was confirmed that the expression was significantly suppressed when the aroma extract was added (ACR-E).

Figure 8 shows the change in FAS gene expression by the aroma extract. FAS is a fatty acid synthetase that is required to accumulate fat in adipocytes, and after the differentiation into adipocytes, the amount of intracellular expression is greatly increased. As shown in Figure 8, when the aroma extract is added, the amount of FAS expression was significantly reduced (ACR-E), it can be seen that the differentiation into adipocytes was suppressed.

Figure 9 shows the changes in FATP-1 gene expression by the aroma extract. FATP-1 is a fatty acid transport protein that helps the absorption of fatty acids to accumulate triglycerides in the liver, adipose tissue or cells. Increased expression of FATP-1 has been reported in diabetic or obese animal models or in fat cells in patients. As shown in Figure 9, after the differentiation into adipocytes its expression amount increased (D), but when treated with aroma extract, the expression amount decreased (ACR-E).

One of the fat cell specific proteins such as LPL and leptin, aP-2, is a fatty acid binding protein in fat cells, which causes lipid droplets to appear in cells.

As shown in FIG. 10, in the differentiated adipocytes (D), gene expression of aP-2 was significantly increased, whereas when treated with aroma extract (ACR-E), it was suppressed to the level before differentiation.

Figure 11 shows the changes in gene expression of SREBP-1 by the aroma extract. SREBP-1 is a transcription factor involved in energy metabolism and lipid synthesis in differentiated adipocytes. It is present in human tissues, liver cells, dermal cells, and adipocytes and is involved in glucose, fatty acid and triglyceride metabolism. It is known that the amount of expression is increased in the diabetes or obesity model. As shown in FIG. 11, SREBP-1 gene expression was increased in adipocytes (D) after differentiation, and when treated with aroma extract (ACR-E), its expression was reduced by 2.5 times as compared to differentiated cells. It became.

Figure 12 shows the gene expression changes of the CTP-1 enzyme, which is involved in the beta oxidation of fatty acids like ACS-1. In particular, the CTP-1 enzyme is an important factor regulating the rate of fatty acid oxidation, and it is known that obesity animals and patients increase the amount of expression in saturated fat cells. As shown in FIG. 12, the expression of CTP-1 was significantly increased in adipocytes (D) after differentiation. However, the expression of CTP-1 was decreased in the case of addition of aroma extract (ACR-E). Could.

As described above, the aroma extract of the present invention suppresses the differentiation into adipocytes without any effect on the survival of adipocytes and effectively reduces the accumulation of lipid droplets (see FIG. 1). In addition, as can be seen in Figures 2 to 12, the gene expression of the transcriptional regulators involved in the degradation / synthesis of glucose, fatty acids, triglycerides and metabolic enzymes expressed under the influence of the aroma extract of the present invention in adipocytes It was confirmed that it is significantly inhibited by. From this, it can be seen that the aroma extract safely and effectively inhibits excessive lipid production, differentiation of fat cells and fat accumulation, and can be used for the treatment and prevention of various diseases related to obesity and excessive lipid production.

Claims (6)

Pharmaceutical composition for the prevention or treatment of diseases selected from the group consisting of obesity, hyperlipidemia and fatty liver containing aroma oil obtained by steam distillation of the aroma as an active ingredient. The composition of claim 1, wherein the aroma is from a plant of the genus Aquilaria . The method of claim 2, wherein the chimhyang Aquila is Ria or cassia (A. khasiana), Aquila Ria Bahia ku Lina (A. apiculina), Aquila in Leah assembly carbonyl (A. blillonil), Aquila Ria bar neon System (A. baneonsis ), Aquila Ria beuraki hits (A. brachyantha), Aquila Ria keonming the ANA (A. cunmingiana), Aquila RIA RIA pillar (A. filaria), Aquila Ria Grandi Flora (A. grandiflora), Aquila Ria Hill rata (a hilata ) , A. microcapa , A. rostrata , A. agallocha , A. malaccensis , A. malaccensis ( A. sinensis ) , or A composition derived from Achilles crassna . Functional food for the prevention or improvement of diseases selected from the group consisting of obesity, hyperlipidemia and fatty liver containing aroma oil obtained by steam distillation of the aroma as an active ingredient. delete delete

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