KR20130103101A

KR20130103101A - Composition for body fat lipolysis using alginate double-layers nanoemulsions containing oleoresin capsicum

Info

Publication number: KR20130103101A
Application number: KR1020120024467A
Authority: KR
Inventors: 김양하; 김종태; 이막순; 김주연; 정선윤
Original assignee: 이화여자대학교 산학협력단; 한국식품연구원
Priority date: 2012-03-09
Filing date: 2012-03-09
Publication date: 2013-09-23

Abstract

The present invention relates to a composition for body fat decomposition of an alginate double-layers nanoemulsion containing an oleoresin capsicum. Furthermore, the present invention comprises a first step of preparing a mixed solution by mixing the oleoresin capsicum and Tween 80; And a second step of preparing the double layers nanoemulsion by mixing the mixed solution and the alginic acid solution.
According to the present invention, a composition for decomposing body fat of an alginate double-layers nanoemulsion containing an oleoresin capsicum and a method for preparing the same can solve the recent health problems caused by overweight and obesity. The beneficial effect is that it is recognized.

Description

Composition for Body Fat Lipolysis Using Alginate Double-layers Nanoemulsions Containing Oleoresin Capsicum}

The present invention relates to a composition for body fat decomposition of an alginate double-layers nanoemulsion containing an oleoresin capsicum.

Furthermore, the present invention comprises a first step of preparing a mixed solution by mixing the oleoresin capsicum and Tween 80; And a second step of preparing the double layers nanoemulsion by mixing the mixed solution and the alginic acid solution.

The rapid change in eating and lifestyle has led to a rapid increase in the prevalence of obesity worldwide, which is becoming a serious health problem. Obesity is known to increase the risk of health problems such as cardiovascular disease, insulin resistance, diabetes, hyperlipidemia, hypertension, and cancer (Park, 2001). Nowadays, overweight, obesity, and related health problems are becoming very common, replacing public health problems such as malnutrition and infectious diseases in the past (World Health Organization, 2000). Obesity is a condition in which fat is excessively accumulated in the body due to an imbalance between energy intake and consumption. Adipose tissue regulates lipid storage and energy metabolism. Obesity causes fat cells to increase or expand in size (Ejaz et al. al ., 2009). In particular, white adipose tissue forms triglycerides as energy storage organs, and releases fatty acids into the bloodstream when energy is scarce (Mercader et. al ., 2006).

According to The Third Korea National Health and Nutrition Examination Survey (KNHANES III, 2005), the prevalence of obesity [BMI≥25.0kg / m ² ] was 31.8% for adults over 20 years of age. Compared to 14.8% in 1995, the prevalence of obesity has more than doubled in the last decade. Therefore, prevention and treatment of obesity are important to reduce the incidence and mortality of diseases (Ejaz et. al ., 2009). Moreover, excessive white adipose tissue causes adipocaine secretion imbalance (Maeda et. al ., 2007; Walker et al ., 2007) Because it is a major cause of obesity and metabolic disorders, it may be an effective way to prevent fat accumulation in preventing it (Joo et. al ., 2010).

Recently, there has been increasing interest in preventing and treating various diseases using functional foods (Crespy et. al ., 2004). In the field of food science, much attention is paid to food ingredients that prevent obesity-related diseases through body fat accumulation inhibition (Lee et al., 2009). In particular, there are several reports on spices used as food additives to improve the taste, aroma and color of food (Kempaiah et. al ., 2006), and many studies indicate that spices inhibit the production of fat cells (Hsu et al ., 2007) or increase energy consumption (Kovacs et. al ., 2006). As such, there is a growing interest in foods that are effective and safe for weight control and obesity treatment.

In the last few years, interest in food nanotechnology has been increasing worldwide. Nanotechnology is a technology that characterizes and controls materials on a nanoscale smaller than 100nm and is considered a potential technology for innovation in agriculture and food systems. Nanoscale structures have unique new functional properties (Weiss et. al ., 2006). For example, food safety (Baeumner, 2004), functional foods for health promotion (Weiss et. al . , 2006), Structural Design Principles for the Delivery of Nanomaterials and Bioactive Components of Food Ingredients (McClements et al ., 2009), Nanotechnology Applications in Food Science and Agriculture (Moraru et. al ., 2003) have been published to address the potential for food nanotechnology. The application of nanotechnology in food and nutrition is to design and develop new functional food ingredients that enhance water solubility, heat resistance, bioavailability, sensory properties and physiological action. Most of the phytochemicals, such as polyphenols, carotenoids, or oleoresin capsicum, are insoluble in solvents or are fat-soluble substances and thus have low oral bioavailability. Therefore, one way to improve the bioavailability of unstable, low solubility functional foods is to use nanoemulsions (Huang et al ., 2010).

Red pepper (capsicum annuum L. ) Is known not only in Korea, but now around a quarter of the world's population eats every day. Red pepper is important for cooking and is widely used as a food, spice, and bad food all over the world. Oleoresin capsicum is obtained by extracting organic solvents from dried berries of red pepper. The bioactive functions of oleoresin capsicum are not yet well known but have been reported to have cholesterol lowering effects (Gupta et al. al ., 2002). To date, no lipolytic effect of oleoresin capsicum is known, and no lipolytic effect of alginic acid bilayer nanoemulsions comprising oleoresin xicum is known. Therefore, the purpose of this study is to compare the biological activities of the lipolytic effect of monolayer nanoemulsion and bilayer nanoemulsion containing oleoresin capsicum.

Hereinafter, the background document of this invention is disclosed.

Baeumner A. Nanosensors identify pathogens in food. Food technol 2004; 58: 51-55

Crespy V, Williamson G. A Review of the Health Effects of Green Tea Catechins in In Vivo Animal Models. J Nutr 2004; 134: 3431S-3440S

Ejaz A, Wu D, Kwan P, Meydani M. Curcumin Inhibits Adipogenesis in 3T3-L1 Adipocytes and Angiogenesis and Obesity in C57 / BL Mice. J Nutr 2009; 139: 919925

Gupta RS, Dixit VP, Dobhal MP. Hypocholesterolaemic effect of the oleoresin of Capsicum annum L. in gerbils (Meriones hurrianae Jerdon). Phytother Res. 2002 May; 16 (3): 273-5.

Hsu CL, Yen GC. Effects of capsaicin on induction of apoptosis and inhibition of adipogenesis in 3T3-L1 cells. J Agric Food Chem 2007; 55: 1730-1736

Huang Q, Yu H, Ru Q. Bioavailability and Delivery of Nutraceuticals Using Nanotechnology. J Food Sci 2010; 75: R50-R57

Joo JI, Kim DH, Choi JW, Yun JW. Proteomic Analysis for Antiobesity Potential of Capsaicin on White Adipocyte Tissue in Rats Fed with High Fat Diet. J Proteome Res 2010; 9: 29772987

Kempaiah RK, Srinivasan K. Beneficial influence of dietary curcumin, capsaicin and garlic on erythrocyte integrity in high-fat fed rats. J Nutr Biochem 2006; 17: 471478

Kovacs EMR, Mela DJ. Metabolically active functional food ingredients for weight control. Obes Rev 2006; 7: 5978

Lee MS, Kim CT, Kim Y. Green Tea () -Epigallocatechin-3-Gallate Reduces Body Weight with Regulation of Multiple Genes Expression in Adipose Tissue of Diet-Induced Obese Mice. Ann Nutr Metab 2009; 54: 151 157

Maeda H, Hosokawa M, Sashima T, Miyashita K. Dietary combination of fucoxanthin and fish oil attenuates the weight gain of white adipose tissue and decreases blood glucose in obese / diabetic KK-Ay mice. J Agric Food Chem 2007; 55: 77017706

McClements DJ, Decker EA, Park Y, Weiss J. Structural Design Principles for Delivery of Bioactive Components in Nutraceuticals and Functional Foods. Crit Rev Food Sci Nutr 2009; 49: 577-606

Mercader J, Ribot J, Murano I, Felipe F, Cinti S, Bonet ML, Palou A. Remodeling of white adipose tissue after retinoic acid administration in mice. Endocrinology 2006; 147: 53255332

The Third Korea National Health and Nutrition Examination Survey (KNHANES III) 2005, Korea Centers for Disease Control and Prevention.

Moraru CI, Panchapakesan CP, Huang QR, Takhistov P, Liu S, Kokini JL. Nanotechnology: A new frontier in food science. Food Technol 2003; 57: 24-29

Park HS. The approach to obesity and medical therapy at primary medical examination. Arch Family Med 2001; 22: 447-458

Walker CG, Zariwala MG, Holness MJ, Sugden MC. Diet, obesity and diabetes: a current update. Clin Sci ( Lond ) 2007; 112: 93111

Weiss J, Takhistov P, Mcclements DJ. Functional Materials in Food Nanotechnology. J Food Sci 2006; 71: R107-R116

World Health Organization. Obesity: Preventing and Managing the Global Epidemic. Report of a WHO Consultation. World Health Organ Tech Rep Ser 2000; 894: i-xii, 1-253.

Recently, in public health, the seriousness of overweight, obesity, etc. rather than malnutrition, infectious diseases, etc. is increasing day by day. One way to solve such a recent public problem has come to invent the functional food of the present invention and its manufacturing method.

On the other hand, the technical problem to be achieved by the present invention is not limited to the above-mentioned technical problems, other technical problems that are not mentioned can be clearly understood by those skilled in the art from the description of the present invention. There will be.

In order to achieve the above object, the present invention provides a composition for body fat decomposition of alginate double-layers nanoemulsion containing oleoresin capsicum (Oleoresin Capsicum).

In the present invention, the first step of preparing a mixed solution by mixing the oleoresin capsicum and Tween 80; And a second step of preparing the double layer nanoemulsion by mixing the mixed solution and the alginic acid solution.

Preferably, the mixing ratio of the mixed solution of the first step may be characterized in that the oleoresin capsicum: Tween 80 = 1: 3.

Preferably, the concentration of the mixed solution of the second step may be characterized in that 0.5% (w / v).

Preferably, after stirring the nanoemulsion, the third step of filtering; may be characterized in that it further comprises.

Preferably, the fourth step of stabilizing at room temperature; may be characterized in that it further comprises.

Preferably, the stirring may be characterized in that 1.9 to 2.1 hours.

Preferably, the room temperature may be characterized in that 15 ~ 25 ℃.

According to the present invention, a composition for decomposing body fat of an alginate double-layers nanoemulsion containing an oleoresin capsicum and a method for preparing the same can solve the recent health problems caused by overweight and obesity. The beneficial effect is that it is recognized.

1 relates to cell viability.
2 relates to lipid accumulation rates.
3 relates to free fatty acids.
4 relates to CEBP-alpha (relative gene expression), PPAR-gamma (relative gene expression), aP2 (relative gene expression).
5 relates to HSL (relative gene expression), CPT-1 alpha (relative gene expression), UCP2 (relative gene expression).
6 is a schematic diagram of the present invention.

The present invention relates to a composition for body fat decomposition of an alginate double-layers nanoemulsion containing oleoresin capsicum.

In another aspect, the present invention comprises the first step of preparing a mixed solution by mixing the oleoresin capsicum and Tween 80; And a second step of preparing the double layers nanoemulsion by mixing the mixed solution and the alginic acid solution.

Preferably, the mixing ratio of the mixed solution of the first step may be characterized in that the oleoresin capsicum: Tween 80 = 1: 3, the concentration of the mixed solution of the second step is 0.5% (w / v) It can be characterized by.

Preferably, the third step of filtering, after stirring the nanoemulsion; may be characterized in that it further comprises, and further comprising a fourth step of stabilizing at room temperature. The stirring may be characterized in that 1.9 to 2.1 hours, the room temperature may be characterized in that 15 to 25 ℃.

Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.

1. Materials and Methods

(1) purchase of materials

The oleoresin capsicum (OC, from Capsicum fruitscens L. , SHU 100,000, India) used in this study was supplied by G & F Co., Ltd. and stored at 4 ℃. Alginate (from Macrocystis pyrifera, medium viscosity, Mw 240,000) was purchased from Sigma (USA).

(2) Nanoemulsion Preparation

Nanoemulsion using alginate was prepared using a magnetic coupling method. The optimal mixing ratio (mass ratio) of oleoresin capsicum and Tween 80 was determined to be 1: 3 by pseudo-ternary phase diagram. Distilled water nanoemusion was prepared as a single layer nanoemulsion, while alginate nanoemulsion was prepared as a double layer. Classified as nanoemulsion. A mixture of oleoresin capsicum and Tween 80 was added to a water and alginic acid solution at a concentration of 0.5% (w / v), respectively, and stirred at room temperature for 2 hours to prepare a single layer and a double layer nanoemulsion. The prepared nanoemulsion was used after filtration at 0.45 μm PVDF filter (Whatman, England) to stabilize at room temperature for 24 hours.

(3) 3T3-L1 adipocyte culture and differentiation induction

Murine 3T3-fat cells were 2 × 10 ³ cells / cm in DMEM culture (Dulbecco's modified Eagle's medium supplemented with 10% newborn calf serum (Life Technologies, MD), 100 mM HEPES, 50 IU penicillin, and 50 μg streptomycin / ml). Plated at a magnification of ² . For the differentiation of 3T3-fat cells, when 3T3-fat cells showed a confluency of about 60%, fat cells were treated with DMEM medium containing 10 μg / ml insulin, 0.25 μM dexamethasone, and 0.5 mM 1-methyl-3-isobutylwanthine for 2 days. Differentiation was induced and then cultured in 10% FBS-DMEM culture for 7 days to confirm the accumulation of fat particles of 80% or more.

(4) Cytotoxicity Assessment

Cytotoxicity of oleoresin capsicum nanoemulsion was measured using CCK-8 kit (Dojindo, Japan). In consideration of the growth rate of each cell, the appropriate number of cells were seeded by floating in a medium containing 10% fetal bovine serum. That is, 3T3-L1 cells were seeded in 96-well plates with 1 × 10 ³ cells and incubated for 24 hours. The oleoresin capsicum nanoemulsion sample, single layer nanoemulsion and alginic acid bilayer nanoemulsion were added at 0.1ng / ml, 1µg / ml, 10µg / ml, 100µg / ml and each experimental group was 96 wells. The same conditions were used for piate and incubated for 48 hours in a 37 ° C., 5% CO ₂ incubator. 10 μl of CCK-solution was added to each well, followed by further incubation for 2 hours, and the absorbance was measured at 450 nm using a microplate reader. Toxicity for each cell was expressed as a percentage of the mean absorbance values obtained from the average absorbance value of each control 3well.

(5) Intracellular Lipid Staining and Quantitation

3T3-L1 adipocytes were washed with PBS and fixed in 10% formalin / PBS pH 7.4. The accumulated fat was then stained using 0.6% Oil Red O. To measure dyed fat content, the solution was dissolved in a solution containing 4% Nonidet P-4 and the absorbance was measured at 520 nm using a spectrophotometer. Each stained fat content was expressed as a percentage of the average absorbance values obtained from the average absorbance value of each control 3 wells.

(6) triglyceride concentration in cells

Intracellular triglyceride concentration was measured using a commercial kit (Asan Pharm, Korea). Protein content for normalization of cell volume was quantified using a bicinchoninic acid (BCA) protein assay (Thermo Scientific, USA) kit.

(7) Real-time quantitative PCR

Total RNA was extracted using TRIzol (Gibco) and the reaction conditions of reverse transcription to synthesize cDNA from the extracted total RNA are as follows. 4 μg total RNA was mixed with 20 μl of total volume containing 5Xbuffer, 1 mM dNTPs, 30 pmole oligo dT19, and 200 U M-MLV (moloney-murine leukemia virus reverse transcriptase, Promega). Reacted. 1 μl of each cDNA was reacted with 2 × SYBR Green PCR master mix (Qiagen) and 0.25 μM primer. Amplification was performed using Rotor-Gene RG-3000A (Corbett Research), and the reaction conditions of real-time PCR were 95 ° C 15 minutes, 95 ° C 15 seconds (denaturation), 56 ° C 30 seconds (annealing), and 72 ° C 30 seconds ( extension). Fluorescence measurements for quantitative analysis were measured at each PCR cycle, and relative quantification of gene expression was performed using the delta-delta Ct method.

(8) Free fatty acid and glycerol determination

Differentiated 3T3-L1 adipocytes were cultured overnight in DMEM containing 2% (wt / vol) fatty acid-free bovine serum albumin (BSA). The next day, the oleoresin capsicum nanoemulsion was treated and incubated for 24 hours. The cultures released from the cells were collected and heat was applied at 65 ° C. for 8 minutes to stop the activity of the enzymes released from the cells. Glycerol content was measured by glycerol analysis kit (Roche Molecular Biochemicals) and free fatty acids were analyzed by acyl-CoA oxidase-based colorimetric kit (NEFA-C; WAKO Pure Chemicals). Protein content was measured and normalized to cell volume.

(9) Statistical Analysis

The results of each experiment were calculated using the SPSS (statistical program) to calculate the mean and standard deviation of the experimental group, and the difference of each analysis item was pre-verified by one-way ANOVA and then P <0.05 level using Duncan's multiple range test. The significance was verified at.

2. Results and discussion

(1) Experimental Example

To determine the lipolytic effect of oleoresin capsicum nanoemulsion, we measured the cytotoxicity of oleoresin capcicom nanoemulsion and measured the fat accumulation inhibitory effect of oleoresin capcicom nanoemulsion. In addition to measuring the fat degradation effect through the analysis of fatty acid and glycerol content, the effects of liposynthesis and lipolysis related gene expression by oleoresin capsicum nanoemulsion were measured and the results were as follows.

(1_1) Cytotoxicity Measurement of Oleoresin Capsicum Nanoemulsion

In order to measure the cytotoxicity of oleoresin capsicum nanoemulsion in 3T3-L1 adipocytes, monolayer nanoemulsion and alginic acid bilayer nanoemulsion were 0.1ng / ml, 1ng / ml, 10ng / ml, 100ng / ml, 1000ng / ml After incubation for 4 hours and 24 hours in each of the culture solution contained in the concentration of was measured using a CCK-8 kit. As a result, both monolayer nanoemulsion and alginic acid bilayer nanoemulsion of oleoresin capsicum did not show cytotoxicity (see FIGS. 1A and 1B).

(1_2) Inhibition of Lipid Accumulation by Oleoresin Capsicum Nanoemulsion

To measure the effect of lipid accumulation by oleoresin capsicum nanoemulsion, monolayer nanoemulsion and alginic acid bilayer nanoemulsion of oleoresin capsicum were applied to 3T3-L1 adipocytes by hour (1 hour, 3 hours, 6 hours, 12 hours, 24 hours). Changes in lipid content in adipocytes over time were analyzed by intracellular fat content by oil-red O staining after 1, 3, 6, 12 and 24 hours incubation with 1000 ng / ml oleoresin capsicum nanoemulsion. Changes in lipid content in adipocytes over time were 5.6%, 5.5%, 10.5%, and 20.2% fat, respectively, compared to monolayer nanoemulsions after 3, 6, 12, and 24 hours of treatment with 1000 ng / ml of alginic acid bilayer nanoemulsions. Intracellular lipid content was reduced (see FIG. 2A).

The triglyceride content in adipocytes by time was also 5.4%, 10.9% and 22.4% in triglyceride content compared to monolayer nanoemulsions when treated with 1000 ng / ml of alginic acid bilayer nanoemulsions for 6, 12 and 24 hours, respectively. This decreased (see Figure 2b).

(1_3) Lipolytic Effects of Oleoresin Capsicum Nanoemulsion

Triglycerides stored in adipocytes are hydrolyzed into free fatty acids and glycerol and released into the blood. To measure the lipolytic effect of oleoresin capsicum nanoemulsion, oleoresin capcicom nanoemulsion was released to cells after treatment with 3T3-L1 adipocytes hourly (1, 3, 6, 12 and 24 hours). The free fatty acid and glycerol contents were measured. Changes in free fatty acid content released into adipocyte cultures by time were 8.4%, 13.6%, and 25.8% of free fatty acids, respectively, when treated with 1000 ng / ml of alginic acid bilayer nanoemulsions for 6, 12, and 24 hours. The content increased (see FIG. 3A). Changes in glycerol content over time also resulted in an increase in glycerol content of 5.7%, 15.2% and 30.32%, respectively, when treated with 1000 ng / ml of alginic acid bilayer nanoemulsions for 6, 12 and 24 hours of incubation (see FIG. 3b). ).

(1_4) Effects of Liposynthesis and Lipolysis-related Gene Expression by Oleoresin Capsicum Nanoemulsion

Changes in the amount of gene expression associated with lipolysis by oleoresin capsicum nanoemulsion in adipocytes were measured. The monolayer nanoemulsion and the alginic acid bilayer nanoemulsion of oleoresin capsicum were treated in different concentrations (0, 1, 10, 100 and 1000 ng / ml) to determine the genes involved in liposynthesis and lipolysis after 24 hours of incubation. Expression levels were compared. Perioxisome proliferator activated receptors-gamma (PPAR-r), CCAAT / enhancer-binding protein-alpha (CEBP-a) and adipocyte fatty acid binding protein (aP2) genes involved in fat accumulation in adipose tissue The expression levels of hormone sensitive lipase (HSL), carnitine palmitoyltransferase-1 (CPT-1) and uncoupling protein-2 (UCP-2) genes were analyzed by real-time PCR. When the alginic acid bilayer nanoemulsion was treated, the expression levels of the liposynthesis related genes PPAR-r, CEBP-a, and aP2 decreased compared to the monolayer nanoemulsion (see FIG. 4), and the lipolysis related genes HSL and CPT-1. And UCP-2 expression increased with increasing concentration (see FIG. 5).

3. Conclusion

The present results suggest that alginate bilayer nanoemulsions containing olegin capsicum inhibit lipid accumulation in adipocytes and promote intracellular triglyceride breakdown to increase free fatty acid and glycerol release from cells. Alginate bilayer nanoemulsion containing olegin capsicum reduces the expression levels of genes PPAR-r, CEBP-a and aP2 involved in monolayer liposynthesis, genes HSL involved in lipolysis, and genes CPT involved in fatty acidization -1 and UCP-2 expression levels were increased. Therefore, the results suggest that alginic acid bilayer nanoemulsion containing olegin capsicum can be used as a novel functional food or medicine useful for body fat breakdown.

Although the present invention has been described in connection with the specific embodiments of the present invention, it is to be understood that the present invention is not limited thereto. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. In addition, the materials of each component described herein can be readily selected and substituted for various materials known to those skilled in the art. Those skilled in the art will also appreciate that some of the components described herein can be omitted without degrading performance or adding components to improve performance. In addition, those skilled in the art may change the order of the method steps described herein depending on the process environment or equipment. Therefore, the scope of the present invention should be determined by the appended claims and equivalents thereof, not by the embodiments described.

Claims

Alginate double-layers nanoemulsion composition for body fat decomposition containing oleoresin capsicum.

A first step of preparing a mixed solution by mixing an oleoresin capsicum and Tween 80; And a second step of preparing the double layer nanoemulsion by mixing the mixed solution and the alginic acid solution.

The method of claim 2, wherein the mixing ratio of the mixed solution of the first step is oleoresin capsicum: Tween 80 = 1: 3.

The method of claim 2, wherein the concentration of the mixed solution of the second step is 0.5% (w / v).

The method of claim 2, further comprising: filtering the nanoemulsion and then filtering the nanoemulsion.

The method of claim 5, further comprising stabilizing at room temperature.

The method of claim 5, wherein the stirring is 1.9 to 2.1 hours.

7. The method of claim 6, wherein the room temperature is 15 to 25 ℃.