WO2009017379A2 - Brine mineral composition for inhibiting differentiation and growth of fat cells - Google Patents

Brine mineral composition for inhibiting differentiation and growth of fat cells Download PDF

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Publication number
WO2009017379A2
WO2009017379A2 PCT/KR2008/004473 KR2008004473W WO2009017379A2 WO 2009017379 A2 WO2009017379 A2 WO 2009017379A2 KR 2008004473 W KR2008004473 W KR 2008004473W WO 2009017379 A2 WO2009017379 A2 WO 2009017379A2
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WIPO (PCT)
Prior art keywords
mineral water
water
mineral
hardness
salt
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PCT/KR2008/004473
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French (fr)
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WO2009017379A4 (en
WO2009017379A3 (en
Inventor
Yong Shik Chu
Yung Geun Yoo
Seon Hwa Kim
Nam Chul Kim
Hae Jin Jang
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Watervis Co., Ltd.
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Publication of WO2009017379A2 publication Critical patent/WO2009017379A2/en
Publication of WO2009017379A3 publication Critical patent/WO2009017379A3/en
Publication of WO2009017379A4 publication Critical patent/WO2009017379A4/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/16—Inorganic salts, minerals or trace elements
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/38—Other non-alcoholic beverages
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/52—Adding ingredients
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/20—Reducing nutritive value; Dietetic products with reduced nutritive value
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/332—Promoters of weight control and weight loss
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2250/00—Food ingredients
    • A23V2250/15—Inorganic Compounds
    • A23V2250/156—Mineral combination
    • A23V2250/16—Potassium
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2250/00—Food ingredients
    • A23V2250/15—Inorganic Compounds
    • A23V2250/156—Mineral combination
    • A23V2250/161—Magnesium
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2250/00—Food ingredients
    • A23V2250/15—Inorganic Compounds
    • A23V2250/156—Mineral combination
    • A23V2250/1614—Sodium

Definitions

  • the present disclosure relates to mineral water containing a unic ⁇ e mineral, and more particularly, to mineral water separated from deep sea water and use thereof.
  • the present disclosure provides mineral water for inhibiting lipid generation and decomposing lipid, the mineral water being preferably separated from deep sea water.
  • the present disclosure also provides a composition containing the mineral water for body fat reduction, weight reduction and/or anti-obesity.
  • the present disclosure also provides a functional food containing the mineral water for inhibiting lipid generation and decomposing lipid.
  • the present disclosure also provides a skin external preparation containing the mineral water for weight reduction.
  • the present disclosure is related to mineral water containing a unic ⁇ e mineral preferably separated from deep sea water, and use thereof.
  • the mineral water can inhibit adipocyte differentiation and thus lipid generation, and accelerate lipolysis of the generated lipid to thereby reduce body fat with the help of abundant mineral components obtained from deep sea water.
  • the mineral water is effective in body fat reduction, weight reduction and/or anti-obesity.
  • the mineral water effective in inhibiting lipid generation and decomposition of the lipid.
  • the mineral water includes water, a magnesium salt, a calcium salt, a potassium salt, and a sodium salt. It is preferable that the mineral water has magnesium:calcium:potassium:sodium content ratio of 3:0.5-1.5:0.5-1.5:0.5-1.5, preferably
  • the magnesium salt may include, for example, magnesium sulfate, magnesium chloride, or a mixture thereof, but it is not limited thereto.
  • the calcium salt may include, for example, calcium sulfate, calcium chloride, or a mixture thereof, but it is not limited thereto.
  • the potassium salt may include, for example, potassium sulfate, potassium chloride, or a mixture thereof, but it is not limited thereto.
  • the sodium salt may include, for example, potassium sulfate, potassium chloride, or a mixture thereof, but it is not limited thereto.
  • Hardness magnesium (mg/L) x 4 + calcium (mg/L) x 2.5 (1)
  • the hardness is 100 or higher, preferably 300 or higher, more preferably 500 or higher.
  • the hardness is 4000 or lower, preferably 3000 or lower, and more preferably 2000 or lower. Consequently, it is preferable that the mineral water has the hardness of 100 to 4000, preferably 300 to 2000, more preferably 500 to 2000.
  • the mineral concentration in the mineral water may be controlled such that it can satisfy the above hardness range.
  • the total mineral concentration in the mineral water is 0.001 to 1 %(w/v), preferably 0.01 to 0.5 %(w/v).
  • the mineral water is preferably obtained from deep sea water.
  • the deep sea water refers to sea water found at approximately 1000 m or deeper below sea level, i.e., a depth where vertical mixing reaction of sea water does not take place during winter. At the depth, organics do not generated through photosynthesis, decomposition is significant, and the effect of chemical or biological hazards and environmental contaminants is quite small. Therefore, big water molecules of very high physicochemical stability can contain a variety of minerals, there. Recently, the deep sea water has been found to have various efficacies, and thus the availability thereof is being increased.
  • the composition ratio of mineral components such as a magnesium salt, a calcium salt, a potassium salt and a sodium salt in the deep sea water is similar to that in the above described mineral water. Therefore, without separate mineral composition control, the mineral water can be easily obtained as follows: mineral components such as a magnesium salt, a calcium salt, a potassium salt and a sodium salt are separated from deep sea water or a filtrate, which is prepared by filtering the deep sea water through a filter of a pore size from 0.1 ⁇ m to 1 ⁇ m, preferably from 0.3 ⁇ m to 0.7 ⁇ m; and the mineral components are dissolved in purified water, wherein the amount of the purified water is controlled such that the mineral water has a hardness in the above described range.
  • the mineral water according to the exemplary embodiment may be the deep sea water itself or a solution obtained by dissolving the mineral components including a magnesium salt, a calcium salt, a potassium salt and a sodium salt separated from the deep sea water in purified water.
  • the deep sea water can be preferably used for the mineral water in that the advantageous characteristics of the deep sea water may also be utilized.
  • a composition for body fat reduction, weight reduction and/or anti-obesity including the mineral water effective in inhibiting lipid generation and decomposing lipid.
  • a functional food for inhibiting lipid generation and decomposing lipid including the above described mineral water.
  • the content of the mineral water in the composition and the functional food can be suitably controlled according to the effects and the forms of the composition and the functional food.
  • the content of the mineral water therein may be 0.01 wt% to 99 wt%, preferably 0.1 wt% to 90 wt%.
  • a skin external preparation for weight reduction including the above described mineral water.
  • the skin external preparation may be the original solution, or a formulation such as dispersion, emulsion, gel, ointment, patch, and the like.
  • the content of the mineral water in the skin external preparation can be suitably controlled according to the effects and the formulations of the skin external preparation.
  • the content of the mineral water therein may be 0.01 wt% to 99 wt%, preferably 0.1 wt% to 90 wt%.
  • the skin external preparation can provide the effect of reducing weight or removing a texture such as cellulite where fat is agglomerated, with the help of the lipid decomposition effect of the mineral water.
  • the mineral water according to the exemplary embodiments can inhibit adipocyte differentiation and thus lipid generation, and accelerate lipolysis of generated lipid, thereby reducing body fat with the help of the abundant mineral components from deep sea water.
  • the mineral water is effective in body fat reduction, weight reduction and/or anti-obesity.
  • FIG. 1 is a graph illustrating a toxicity of a mineral water according to an exemplary embodiment, according to hardness of the mineral water.
  • FIG. 2 is a graph illustrating a fat cell generation rate of a mineral water according to an exemplary embodiment, according to hardness of the mineral water.
  • FIG. 3 is a graph illustrating an inhibitory effect of a mineral water according to an exemplary embodiment, on adipocyte differentiation according to hardness of the mineral water.
  • FIG. 4 is a graph illustrating an inhibitory effect of a mineral water according to an exemplary embodiment, on adipocyte differentiation after a differentiation accelerator treatment according to hardness of the mineral water.
  • FIG. 5 is a graph illustrating an inhibitory effect of a mineral water according to an exemplary embodiment, on expression of a gene inducing a fat cell treated with a differential accelerator according to hardness of the mineral water.
  • FIG. 6 is a graph illustrating an effect of a mineral water according to an exemplary embodiment, on a body weight according to time and hardness of the mineral water.
  • FIG. 7 is a graph illustrating an effect of a mineral water according to an exemplary embodiment, on a body fat weight according to hardness of the mineral water.
  • FIG. 8 is a graph illustrating an effect of a skin preparation containing a mineral water according to an exemplary embodiment, on reduction of a subcutaneous fat and a cellulite according to hardness of the mineral water.
  • the present disclosure provides mineral water containing a unic ⁇ e mineral, a composition containing the mineral water for body fat reduction, weight reduction and/ or anti-obesity, a functional food containing the mineral water for inhibiting lipid generation and accelerating lipid decomposition, and a skin external preparation containing the mineral water for weight reduction and removal of the lipid texture.
  • the mineral water contains water, a magnesium salt, a calcium salt, a potassium salt and a sodium salt.
  • the content ratio of magnesium saltcalcium saltpotassium saltsodium salt is preferably 3:1:1:1.
  • the hardness of the mineral water is preferably from 500 to 2000 as calculated according to the following e ⁇ ation:
  • Hardness magnesium (mg/L) x 4 + calcium (mg/L) x 2.5.
  • the total mineral concentration in the mineral water is preferably 0.01 to 0.5 %(w/v).
  • Deep sea water which is sea water found at approximately 1000 m or deeper below sea level, contains a variety of minerals.
  • the inventors found that the content ratio of the mineral components including the magnesium salt, the calcium salt, the potassium salt and the sodium salt in the deep sea water is similar to the above described content ratio in the mineral water of the present invention. Therefore, the mineral water of the present invention preferably includes the deep sea water. Mode for the Invention
  • Deep sea water (Tonghae and Yangyang, Korea) was thoroughly filtered through a micro-filter (material: polytetrafluoroethylene (PTFE), pore size: approximately 0.5 ⁇ m, Saehan Co., Ltd.) to remove impurities (pretreated water).
  • the pretreated water was separated into fresh water and concentrate water using a reverse osmosis system (Dow Chemical Company, FILMTEC, SW30-4021, yield: 0.5).
  • Table 1 shows flow rates (unit: gallon per day (GPD)) and mineral concentrations of the deep sea water, the pretreated water and the concentrate water. Baume degree of the concentrate water was 4.5 0 Be.
  • the concentrate water sequentially passed through three multipurpose evaporators to have the Baume degree of 23 0 Be, 30 0 Be, and 36 0 Be to thereby remove calcium salts, sodium salts and sulfates, respectively. Then, the concentrate water was placed in an evapocrystallkation device and concentrated at a temperature of 50 0 C under a pressure of 15 mmHg to obtain a mixed salt slurry of potassium salts and magnesium salts. The slurry was inserted in a washing column and washed with water to obtain a solution containing dissolved magnesium salts and a slurry containing potassium chloride (KCl) crystals. The potassium chloride (KCl) slurry was centrifuged and dried to obtain solid state potassium chloride crystals.
  • KCl potassium chloride
  • the solution containing dissolved magnesium salts was dehydrated and concentrated in a concentration device to precipitate mixed crystals of potassium salts and magnesium salts and sodium chloride crystals and obtain a mixed solution of magnesium chloride hexahydrate (MgCl -H O)
  • magnesium sulfate heptahydrate (MgSO -H O) of an improved magnesium purity.
  • the concentration of magnesium salts was 35.2 wt%.
  • the content ratio of magnesium chloride:magnesium sulfate in the final magnesium salt solution was 4-7:1.
  • the concentration of other inorganic salts was approximately 3.2 wt%, indicating a magnesium solution of an improved purity.
  • MgCl magnesium sulfate
  • MgSO magnesium sulfate
  • KCl - p L otassium chloride
  • NaCl sodium chloride
  • Hardness magnesium (mg/L) x 4 + calcium (mg/L) x 2.5 (1)
  • 3T3-L1 cells (ATCC: CL-173, 2xlO 5 cell/6 well), which are preadipocytes, were seeded in culture dishes.
  • the cells were fed with DMEM (Dulbecco's modified eagle's medium) containing penicillin (100 IU/m-6) and 10% FBS (fetal bovine serum) or other medium providing equivalent or better culture condition.
  • DMEM Dulbecco's modified eagle's medium
  • penicillin 100 IU/m-6
  • FBS fetal bovine serum
  • the culture dishes were maintained at 37 0 C for 48 hours in an incubator containing 5 % carbon dioxide.
  • MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide) analysis was performed to determine the cytotoxicity of the samples.
  • the mineral water of a hardness 4000, prepared from Preparation 2 was used as it was or after being diluted to hardnesses 100, 300, 500, 1000, 2000 and 3000, respectively.
  • the cells were cultured for 48 hours.
  • the cells were cultured further for 4 hours.
  • each was added with 100 j ⁇ of dimethylsulf oxide solution and shaken for 10 minutes. Thereafter, the cell viability was calculated by reading the absorbance at 490 nm from ELISA reader. The results are shown in FIG. 1.
  • 3T3-L1 cells (2x10 cell/well) were cultured in DMEM containing 10% fetal bovine serum (FBS), 4.5 g/£ glucose, 2 rnM glutamine, and antibiotic penicilin- streptomycin (Gibco) at 37 0 C until the cell is completely grown. After that, they were cultured in DMEM added with insulin (10 ⁇ g/m#, Sigma), 0.25 ⁇ M dexamethasone (Sigma), and 0.5 mM isobutylmethylxanthine (Sigma) at 37 0 C for 2 days. They were cultured further in media added with only insulin (10 ⁇ g/m#) at 37 0 C for 3 days. Then, the media were replaced with DMEMFBS added with insulin, dexamethasone, and isobutylmethylxanthine every two days until the cells were used in the experiment.
  • FBS fetal bovine serum
  • Gibco antibiotic penicilin- streptomycin
  • the differentiated cells were used when 90 % or more of the cells showed an adipocyte phenotype by accumulation of lipid droplets.
  • DMEM powder was dissolved in mineral water of a hardness 4000 to prepare DMEM of a hardness 4000. Then, they were diluted with DMEM of a hardness 0 to prepare groups of hardnesses 100, 300, 500, 1000, 2000 and 3000, respectively.
  • a group of a hardness 4000 was prepared using DMEM of a hardness 4000.
  • the cells cultured in 6-wells were treated with 1 ml of mineral water diluted with DMEM to hardnesses 100, 300, 500, 1000, 2000, 3000 and 4000, respectively. After 48 hours, the lipid droplets in the cells were stained with 10% oil red O, extracted with ethanol, and ⁇ antified at 515 nm. The results are shown in FIG. 2.
  • the group of a hardness approximately 100 showed approximately 10 % or higher inhibition against lipid generation
  • the groups of a hardness 300 or higher showed 20 % or higher (approximately 25 % to approximately 45 %) inhibition against lipid generation.
  • 3T3-L1 cells (2x10 cell/well) were cultured in DMEM containing 10% fetal bovine serum (FBS), 4.5 g/£ glucose, 2 mM glutamine, and antibiotic at 37 0 C until the cell is completely grown. After that, they were cultured in DMEMFBS (the medium was prepared as described above) added with insulin (10 ⁇ g/m-6), 0.25 ⁇ M dexamethasone, 0.5 mM isobutylmethylxanthine, and mineral water diluted with DMEM to hardnesses 100, 300, 500, 1000, 2000, 3000 and 4000, respectively, at 37 0 C for 2 days.
  • FBS fetal bovine serum
  • DMEMFBS the medium was prepared as described above
  • insulin 10 ⁇ g/m-6
  • 0.25 ⁇ M dexamethasone 0.5 mM isobutylmethylxanthine
  • mineral water diluted with DMEM to hardnesses 100, 300, 500, 1000, 2000, 3000 and 4
  • the group of a hardness 1000 showed approximately 35 % inhibition against adipocyte differentiation
  • the group of a hardness 2000 showed approximately 40 % inhibition against adipocyte differentiation.
  • 3T3-L1 cells (2x10 cell/well) were cultured in DMEM containing 10% fetal bovine serum (FBS), 4.5 g/£ glucose, 2 mM glutamine, and antibiotic. After completely grown, the cells were cultured in DMEMFBS (prepared as described above) added with insulin (10 ⁇ g/m#), 0.25 ⁇ M dexamethasone, 0.5 mM isobutylmethylxanthine, 10 ⁇ M troglitazone (Sigma) which is an accelerator for adipocyte differentiation, and mineral water diluted with DMEM to hardnesses 100, 300, 500, 1000, 2000, 3000 and 4000, respectively, at 37 0 C for 2 days.
  • FBS fetal bovine serum
  • DMEMFBS prepared as described above
  • insulin 10 ⁇ g/m#
  • 0.25 ⁇ M dexamethasone 0.5 mM isobutylmethylxanthine
  • 10 ⁇ M troglitazone (Sigma) which is an accelerator for
  • the cells were cultured further in media added with only insulin (10 ⁇ g/m#) at 37 0 C for 3 days. Then, the media were replaced with DMEMFBS added with insulin, dexamethasone, isobutylmethylxanthine, and troglitazone every two days until the cells were used in the experiment.
  • the cells were treated with mineral water diluted with DMEM to hardnesses 100, 300, 500, 1000, 2000, 3000 and 4000, respectively. After 48 hours at 37 0 C, the lipid droplets in the cells were stained with 10% oil red O, extracted with ethanol, and quantified at 515 nm.
  • FIG. 4 The effect of the mineral water on inhibiting the adipocyte differentiation after the differentiation accelerator treatment was performed is shown in FIG. 4 according to the hardness. As shown in FIG. 4, after 5 days, the groups of a hardness 500 or higher showed 10 % or higher (approximately 10 % to approximately 40 %) inhibition against lipid generation.
  • Adipocyte While the differentiation of the preadipocyte to the adipocyte is induced by insulin, transcriptional expression of transcriptional factors such as PPAR ⁇ , C/EBP famil, ADD1/SREBP1 and the like increase. In this experiment, so as to investigate the effect of the mineral water on inhibiting expression of genes related to the adipocyte induction, the expression of the genes in a cell was analyzed using PPAR ⁇ through real time PCR.
  • 3T3-L1 cells (6xlO 5 cell / 25 cm 2 T-flask) were cultured in DMEM containing 10% fetal bovine serum (FBS), 4.5 g/£ glucose, 2 mM glutamine, and antibiotic. After completely grown, the cells were cultured in DMEMFBS added with insulin (10 ⁇ g/ m#), 0.25 ⁇ M dexamethasone, 0.5 mM isobutylmethylxanthine, 10 ⁇ M troglitazone (Sigma) which is an accelerator for adipocyte differentiation, and mineral water diluted with DMEM to hardnesses 1000, 2000 and 4000, respectively, at 37 0 C for 2 days.
  • FBS fetal bovine serum
  • DMEMFBS fetal bovine serum
  • insulin 10 ⁇ g/ m#
  • 0.25 ⁇ M dexamethasone 0.5 mM isobutylmethylxanthine
  • 10 ⁇ M troglitazone (Sigma) which is an accelerator for
  • RNA were ⁇ antified using Genensis 10 spectrophotometer (Thermo scientific, USA).
  • FIG. 5 The effect of the mineral water on inhibiting the expression of the genes inducing adipocyte after treated with the accelerator for adipocyte differentiation is shown in FIG. 5. As shown in FIG. 5, as the hardness increases, the expression of PPAR ⁇ was inhibited, and the mineral water of a hardness 1000 showed 50 % inhibition against gene expression, the mineral water of a hardness 2000 showed 67 % inhibition against gene expression, and the mineral water of a hardness 4000 showed 95 % inhibition against gene expression, providing significant results.
  • 80+10 g were purchased. They were fed with cubed diets (Samyang Feed, Korea) and free water in an animal laboratory for 7 days (adaptation period). After the adaptation period, they were divided into 6 groups including a group fed with distilled water, and 5 groups fed with mineral water of hardnesses 100, 500, 1000, 2000, and 4000, respectively. The 6 groups of the white rats were fed with corresponding cubed diets and free water for 5 weeks (breeding period). During the breeding period, their weight variation and amount of water intake were measured. Thereafter, obesity inhibition experiments were performed on them. The animal room was maintained at a constant temperature and humidity condition (23+ 3 0 C, 50+ 10% RH), and light was automatically controlled at 12 hours' intervals from 08: 00 to 20: 00.
  • the mineral water of a hardness 4000 prepared in Preparation 2 was used without dilution, or after being diluted with distilled water to have hardnesses 100, 500, 1000 and 2000, respectively.
  • Every white rat was measured for body weight at 2 o'clock every two days.
  • the white rats were fed with weighted diets, and the remaining water was measured the next day to calculate the amount of daily water intake.
  • the rats were anesthetized.
  • the weights of the brown adipose fat (BAT) and the epididymal fat were measured before the perfusion, and the weights of the peritoneal fat and the visceral fat were measured after the perfusion, to compare the weights of the body fats for respective regions.
  • the distilled water group (the group of white rats fed with distilled water) and the mineral water groups (the groups of white rats fed with mineral water of hardnesses 100, 500, 1000, 2000 and 4000, respectively) were measured for variations of body weights according to the water intake for 3 weeks, and the results are shown in FIG. 6. As shown in FIG. 6, all of the mineral water groups showed significant effect of inhibiting body weight increase. The time “0" in FIG. 6 indicates the treatment time of the mineral water. From the comparison of the final body weights after the treatment, it was found that the mineral water groups had a significant inhibitory effect on the increase of the body weight, and 5 % to 25 % of the body weight was decreased for 3 weeks.
  • Mineral water skin preparation was prepared using the mineral water according to the exemplary embodiment, and their compositions are listed in Table 2.
  • the mineral water includes four groups of mineral waters having hardnesses 100, 1000, 2000 and 4000, respectively.
  • the mineral water of a hardness 4000 prepared in Preparation 2 was used without dilution, or after being diluted to hardnesses 100, 1000 and 2000 with distilled water.
  • the deep sea mineral water among the materials for the cosmetics may be referred to as 'sea water' regardless of the specifications.
  • BMI body mass index
  • the girth of the thigh was found to be 0.3 cm to 0.6 cm smaller in the inventive examples (applied with the skin preparation containing the mineral water) than in the comparative example (applied with the skin preparation containing the distilled water), providing a statistically significant result.
  • the body weight of the subject did not varied during the experiment.
  • FIG. 8 is a graph illustrating reduction rates of the subcutaneous fat and the cellulite after the application of the skin preparation containing the mineral water according to the exemplary embodiment.
  • the thigh applied with the skin preparation containing the mineral water had the subcutaneous fat reduced by 2.7 % compared to the state before the application (in the case of a hardness 100), providing a statistically significant result.
  • the comparative example did not show a significant variation.
  • the inventive examples e.g., the case where the mineral water has a hardness of 1000 showed a cellulite reduction rate of approximately 15 %, providing a statistically significant result.
  • mineral water containing a unique mineral particularly, mineral water separated from deep sea water, and use thereof.
  • the deep sea water composition is effective in inhibiting lipid generation with the help of the mineral components contained therein and thus can be applied to beverage and food compositions and skin cosmetics for anti-obesity and weight reduction.

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Abstract

The mineral water inhibits adipocyte differentiation and thus generation of lipids and accelerates lipolysis of generated lipid, thereby reducing body fat with the help of abundant mineral components from deep sea water. Hence, the mineral water is effective in body fat reduction, weight reduction and/or anti-obesity.

Description

Description
BRINE MINERAL COMPOSITION FOR INHIBITING DIFFERENTIATION AND GROWTH OF FAT CELLS
Technical Field
[1] The present disclosure relates to mineral water containing a unicμe mineral, and more particularly, to mineral water separated from deep sea water and use thereof. Background Art
[2] Recently, as total caloric intake increases, and particularly fat intake increases, the rate of obesity increases every year. The obesity is a state where excess body fat is accumulated based on supernutrition. When energy intake exceeds energy expenditure, the excess energy is accumulated in a body as neutral fat (body fat) by a white fat cell, which is a cause of the obesity. The obesity caused by the accumulation of the body fat is not good for beauty, and is a cause of various diseases such as hyperlipidemia.
[3] In order to prevent such diseases and reduce weight, researches have been made to treat the obesity using a variety of foods such as dietary fiber, hot water extract of Cnidium officinale, cocoon, and Lycinum chineuse.
[4] However, most weight reduction and anti-obesity preparations yet developed are not suitable to be used generally because of their peculiar flavors offending some users.
[5] Therefore, the inventors have conducted researches for developing a preparation that has superior general-use characteristics and is effective in body fat reduction, weight reduction and/or anti-obesity without peculiar flavor or taste. Disclosure of Invention Technical Problem
[6] The present disclosure provides mineral water for inhibiting lipid generation and decomposing lipid, the mineral water being preferably separated from deep sea water.
[7] The present disclosure also provides a composition containing the mineral water for body fat reduction, weight reduction and/or anti-obesity.
[8] The present disclosure also provides a functional food containing the mineral water for inhibiting lipid generation and decomposing lipid.
[9] The present disclosure also provides a skin external preparation containing the mineral water for weight reduction. Technical Solution
[10] The present disclosure is related to mineral water containing a unicμe mineral preferably separated from deep sea water, and use thereof. The mineral water can inhibit adipocyte differentiation and thus lipid generation, and accelerate lipolysis of the generated lipid to thereby reduce body fat with the help of abundant mineral components obtained from deep sea water. Hence, the mineral water is effective in body fat reduction, weight reduction and/or anti-obesity.
[11] In accordance with an exemplary embodiment, there is provided mineral water effective in inhibiting lipid generation and decomposition of the lipid. The mineral water includes water, a magnesium salt, a calcium salt, a potassium salt, and a sodium salt. It is preferable that the mineral water has magnesium:calcium:potassium:sodium content ratio of 3:0.5-1.5:0.5-1.5:0.5-1.5, preferably
3:0.75-1.25:0.75-1.25:0.75-1.25, more preferably approximately 3:1:1:1 by weight, which is similar to a mineral composition of an organism, preferably to a human body. The magnesium salt may include, for example, magnesium sulfate, magnesium chloride, or a mixture thereof, but it is not limited thereto. The calcium salt may include, for example, calcium sulfate, calcium chloride, or a mixture thereof, but it is not limited thereto. The potassium salt may include, for example, potassium sulfate, potassium chloride, or a mixture thereof, but it is not limited thereto. The sodium salt may include, for example, potassium sulfate, potassium chloride, or a mixture thereof, but it is not limited thereto.
[12] Hardness of the mineral water can be calculated according to the following eψation:
[13] Hardness = magnesium (mg/L) x 4 + calcium (mg/L) x 2.5 (1)
[14] If the hardness is too low, the effect of the mineral water on accelerating lipid decomposition and inhibiting lipid generation is quite small. Therefore, in order to obtain sufficient effect, it is preferable that the hardness is 100 or higher, preferably 300 or higher, more preferably 500 or higher. However, if the hardness is too high, side effects such as cytotoxicity may be caused. Therefore, in order to minimize the cytotoxicity, it is preferable that the hardness is 4000 or lower, preferably 3000 or lower, and more preferably 2000 or lower. Consequently, it is preferable that the mineral water has the hardness of 100 to 4000, preferably 300 to 2000, more preferably 500 to 2000.
[15] The mineral concentration in the mineral water may be controlled such that it can satisfy the above hardness range. For example, in order to satisfy the above hardness range and realize a condition similar to the body mineral environment, the total mineral concentration in the mineral water is 0.001 to 1 %(w/v), preferably 0.01 to 0.5 %(w/v). [16] The mineral water is preferably obtained from deep sea water. The deep sea water refers to sea water found at approximately 1000 m or deeper below sea level, i.e., a depth where vertical mixing reaction of sea water does not take place during winter. At the depth, organics do not generated through photosynthesis, decomposition is significant, and the effect of chemical or biological hazards and environmental contaminants is quite small. Therefore, big water molecules of very high physicochemical stability can contain a variety of minerals, there. Recently, the deep sea water has been found to have various efficacies, and thus the availability thereof is being increased.
[17] The inventors found that the composition ratio of mineral components such as a magnesium salt, a calcium salt, a potassium salt and a sodium salt in the deep sea water is similar to that in the above described mineral water. Therefore, without separate mineral composition control, the mineral water can be easily obtained as follows: mineral components such as a magnesium salt, a calcium salt, a potassium salt and a sodium salt are separated from deep sea water or a filtrate, which is prepared by filtering the deep sea water through a filter of a pore size from 0.1 μm to 1 μm, preferably from 0.3 μm to 0.7 μm; and the mineral components are dissolved in purified water, wherein the amount of the purified water is controlled such that the mineral water has a hardness in the above described range.
[18] Therefore, the mineral water according to the exemplary embodiment may be the deep sea water itself or a solution obtained by dissolving the mineral components including a magnesium salt, a calcium salt, a potassium salt and a sodium salt separated from the deep sea water in purified water. As described above, the deep sea water can be preferably used for the mineral water in that the advantageous characteristics of the deep sea water may also be utilized.
[19] In accordance with another exemplary embodiment, there is provided a composition for body fat reduction, weight reduction and/or anti-obesity, including the mineral water effective in inhibiting lipid generation and decomposing lipid. In accordance with still another exemplary embodiment, there is provided a functional food for inhibiting lipid generation and decomposing lipid, including the above described mineral water. The content of the mineral water in the composition and the functional food can be suitably controlled according to the effects and the forms of the composition and the functional food. For example, the content of the mineral water therein may be 0.01 wt% to 99 wt%, preferably 0.1 wt% to 90 wt%.
[20] In accordance with further another exemplary embodiment, there is provided a skin external preparation for weight reduction, including the above described mineral water. The skin external preparation may be the original solution, or a formulation such as dispersion, emulsion, gel, ointment, patch, and the like. The content of the mineral water in the skin external preparation can be suitably controlled according to the effects and the formulations of the skin external preparation. For example, the content of the mineral water therein may be 0.01 wt% to 99 wt%, preferably 0.1 wt% to 90 wt%. As applied to the skin, the skin external preparation can provide the effect of reducing weight or removing a texture such as cellulite where fat is agglomerated, with the help of the lipid decomposition effect of the mineral water.
Advantageous Effects
[21] The mineral water according to the exemplary embodiments can inhibit adipocyte differentiation and thus lipid generation, and accelerate lipolysis of generated lipid, thereby reducing body fat with the help of the abundant mineral components from deep sea water. Hence, the mineral water is effective in body fat reduction, weight reduction and/or anti-obesity. Brief Description of the Drawings
[22] FIG. 1 is a graph illustrating a toxicity of a mineral water according to an exemplary embodiment, according to hardness of the mineral water.
[23] FIG. 2 is a graph illustrating a fat cell generation rate of a mineral water according to an exemplary embodiment, according to hardness of the mineral water.
[24] FIG. 3 is a graph illustrating an inhibitory effect of a mineral water according to an exemplary embodiment, on adipocyte differentiation according to hardness of the mineral water.
[25] FIG. 4 is a graph illustrating an inhibitory effect of a mineral water according to an exemplary embodiment, on adipocyte differentiation after a differentiation accelerator treatment according to hardness of the mineral water.
[26] FIG. 5 is a graph illustrating an inhibitory effect of a mineral water according to an exemplary embodiment, on expression of a gene inducing a fat cell treated with a differential accelerator according to hardness of the mineral water.
[27] FIG. 6 is a graph illustrating an effect of a mineral water according to an exemplary embodiment, on a body weight according to time and hardness of the mineral water.
[28] FIG. 7 is a graph illustrating an effect of a mineral water according to an exemplary embodiment, on a body fat weight according to hardness of the mineral water.
[29] FIG. 8 is a graph illustrating an effect of a skin preparation containing a mineral water according to an exemplary embodiment, on reduction of a subcutaneous fat and a cellulite according to hardness of the mineral water.
Best Mode for Carrying Out the Invention
[30] The present disclosure provides mineral water containing a unicμe mineral, a composition containing the mineral water for body fat reduction, weight reduction and/ or anti-obesity, a functional food containing the mineral water for inhibiting lipid generation and accelerating lipid decomposition, and a skin external preparation containing the mineral water for weight reduction and removal of the lipid texture.
[31] The mineral water contains water, a magnesium salt, a calcium salt, a potassium salt and a sodium salt. The content ratio of magnesium saltcalcium saltpotassium saltsodium salt is preferably 3:1:1:1. The hardness of the mineral water is preferably from 500 to 2000 as calculated according to the following eψation:
[32] Hardness = magnesium (mg/L) x 4 + calcium (mg/L) x 2.5.
[33] The total mineral concentration in the mineral water is preferably 0.01 to 0.5 %(w/v).
[34] Deep sea water, which is sea water found at approximately 1000 m or deeper below sea level, contains a variety of minerals. The inventors found that the content ratio of the mineral components including the magnesium salt, the calcium salt, the potassium salt and the sodium salt in the deep sea water is similar to the above described content ratio in the mineral water of the present invention. Therefore, the mineral water of the present invention preferably includes the deep sea water. Mode for the Invention
[35] Hereinafter, specific embodiments will be described in detail with reference to the accompanying drawings. The present invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the present invention to those skilled in the art.
[36]
[37] EXAMPLES
[38] Preparation 1 : Extraction and Purification of Deep Sea Water
[39] Deep sea water (Tonghae and Yangyang, Korea) was thoroughly filtered through a micro-filter (material: polytetrafluoroethylene (PTFE), pore size: approximately 0.5 μm, Saehan Co., Ltd.) to remove impurities (pretreated water). The pretreated water was separated into fresh water and concentrate water using a reverse osmosis system (Dow Chemical Company, FILMTEC, SW30-4021, yield: 0.5). Table 1 shows flow rates (unit: gallon per day (GPD)) and mineral concentrations of the deep sea water, the pretreated water and the concentrate water. Baume degree of the concentrate water was 4.5 0Be.
[40] Table 1 [Table 1] [Table ]
Figure imgf000007_0001
[41] The concentrate water sequentially passed through three multipurpose evaporators to have the Baume degree of 23 0Be, 30 0Be, and 36 0Be to thereby remove calcium salts, sodium salts and sulfates, respectively. Then, the concentrate water was placed in an evapocrystallkation device and concentrated at a temperature of 50 0C under a pressure of 15 mmHg to obtain a mixed salt slurry of potassium salts and magnesium salts. The slurry was inserted in a washing column and washed with water to obtain a solution containing dissolved magnesium salts and a slurry containing potassium chloride (KCl) crystals. The potassium chloride (KCl) slurry was centrifuged and dried to obtain solid state potassium chloride crystals. The solution containing dissolved magnesium salts was dehydrated and concentrated in a concentration device to precipitate mixed crystals of potassium salts and magnesium salts and sodium chloride crystals and obtain a mixed solution of magnesium chloride hexahydrate (MgCl -H O)
2 2 and magnesium sulfate heptahydrate (MgSO -H O) of an improved magnesium purity.
4 2
In the final magnesium salt solution, the concentration of magnesium salts was 35.2 wt%. For reference, the content ratio of magnesium chloride:magnesium sulfate in the final magnesium salt solution was 4-7:1. The concentration of other inorganic salts was approximately 3.2 wt%, indicating a magnesium solution of an improved purity.
[42] [43] Preparation 2: Preparation of Mineral Water
[44] 267.88 mg of calcium sulfate (CaSO ), 1172 mg of magnesium chloride (MgCl ) and
4 2 magnesium sulfate (MgSO ) (corresponding to 976.7 mg of magnesium chloride
4
(MgCl ) and 195.3 mg of magnesium sulfate (MgSO ) according to the content ratio MgCl 2 :MgSO 4 =5:1), 150.43 mg c of - pL otassium chloride (KCl), 200.43 mg c of sodium chloride (NaCl) were solved in 0.522 L permeated water and agitated to prepare mineral water having a weight ratio of MgCa:K:Na = 3:1:1:1, which is similar to that of minerals in human body.
[45] Hardness of the mineral water was calculated according to the following equation:
[46] Hardness = magnesium (mg/L) x 4 + calcium (mg/L) x 2.5 (1)
[47] Hardness of the mineral water calculated from Equation 1 was 4000. All the mineral water used in the following experiments were appropriately diluted according to the desired hardness before the experiments.
[48]
[49] Experiment 1: Measurement of Cytotoxicity According to Hardness
[50] 3T3-L1 cells (ATCC: CL-173, 2xlO5 cell/6 well), which are preadipocytes, were seeded in culture dishes. The cells were fed with DMEM (Dulbecco's modified eagle's medium) containing penicillin (100 IU/m-6) and 10% FBS (fetal bovine serum) or other medium providing equivalent or better culture condition. The culture dishes were maintained at 37 0C for 48 hours in an incubator containing 5 % carbon dioxide. MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide) analysis was performed to determine the cytotoxicity of the samples.
[51] The procedure for determining the cytotoxicity (MTT analysis) was as follows.
[52] The mineral water of a hardness 4000, prepared from Preparation 2, was used as it was or after being diluted to hardnesses 100, 300, 500, 1000, 2000 and 3000, respectively. After adding 0.05 ml of each of the mineral water and 0.05 ml of cell culture solution in corresponding well, the cells were cultured for 48 hours. Then, after adding 0.5 % MTT solution in each well, the cells were cultured further for 4 hours. After removing the culture solution, each was added with 100 jΛ of dimethylsulf oxide solution and shaken for 10 minutes. Thereafter, the cell viability was calculated by reading the absorbance at 490 nm from ELISA reader. The results are shown in FIG. 1.
[53] As shown in FIG. 1, the groups treated with up to 25 % of the mineral water
(hardness: 1000) showed nontoxicity similar to the untreated group (hardness: 0).
[54]
[55] Experiment 2: Inhibitory Effect of Mineral Water on Lipid Generation in 3T3-L1 Adipocytes
[56] 3T3-L1 cells (2x10 cell/well) were cultured in DMEM containing 10% fetal bovine serum (FBS), 4.5 g/£ glucose, 2 rnM glutamine, and antibiotic penicilin- streptomycin (Gibco) at 37 0C until the cell is completely grown. After that, they were cultured in DMEM added with insulin (10 μg/m#, Sigma), 0.25 μM dexamethasone (Sigma), and 0.5 mM isobutylmethylxanthine (Sigma) at 37 0C for 2 days. They were cultured further in media added with only insulin (10 μg/m#) at 37 0C for 3 days. Then, the media were replaced with DMEMFBS added with insulin, dexamethasone, and isobutylmethylxanthine every two days until the cells were used in the experiment.
[57] The differentiated cells were used when 90 % or more of the cells showed an adipocyte phenotype by accumulation of lipid droplets. Specifically, DMEM powder was dissolved in mineral water of a hardness 4000 to prepare DMEM of a hardness 4000. Then, they were diluted with DMEM of a hardness 0 to prepare groups of hardnesses 100, 300, 500, 1000, 2000 and 3000, respectively. A group of a hardness 4000 was prepared using DMEM of a hardness 4000. The cells cultured in 6-wells were treated with 1 ml of mineral water diluted with DMEM to hardnesses 100, 300, 500, 1000, 2000, 3000 and 4000, respectively. After 48 hours, the lipid droplets in the cells were stained with 10% oil red O, extracted with ethanol, and ψantified at 515 nm. The results are shown in FIG. 2.
[58] As shown in FIG. 2, the group of a hardness approximately 100 showed approximately 10 % or higher inhibition against lipid generation, and the groups of a hardness 300 or higher showed 20 % or higher (approximately 25 % to approximately 45 %) inhibition against lipid generation.
[59]
[60] Experiment 3: Inhibitory Effect of Mineral Water on Differentiation of 3T3-L1 Cell
(Preadipocyte) to Adipocyte
[61] 3T3-L1 cells (2x10 cell/well) were cultured in DMEM containing 10% fetal bovine serum (FBS), 4.5 g/£ glucose, 2 mM glutamine, and antibiotic at 37 0C until the cell is completely grown. After that, they were cultured in DMEMFBS (the medium was prepared as described above) added with insulin (10 μg/m-6), 0.25 μM dexamethasone, 0.5 mM isobutylmethylxanthine, and mineral water diluted with DMEM to hardnesses 100, 300, 500, 1000, 2000, 3000 and 4000, respectively, at 37 0C for 2 days. They were cultured further in media added with only insulin (10 μg/m#) and sample at 37 0C for 3 days. Then, the media were replaced with DMEMFBS added with insulin, dexamethasone, isobutylmethylxanthine, and the sample every two days until the cells were used in the experiment. They were analyzed when 90 % or more of the cells in a control group added with no mineral water showed an adipocyte phenotype by accumulation of lipid droplets. The lipid droplets in the cells were stained with 10% oil red O, extracted with ethanol, and ψantified at 515 nm. The results are shown in FIG. 3.
[62] As shown in FIG. 3, the groups of hardnesses 100 to 300 had no significant effect.
However, the group of a hardness 1000 showed approximately 35 % inhibition against adipocyte differentiation, and the group of a hardness 2000 showed approximately 40 % inhibition against adipocyte differentiation.
[63]
[64] Experiment 4: Inhibitory Effect of Mineral Water on Differentiation of 3T3-L1 Cell
(Preadipocyte) Treated with Differentiation Accelerator to Adipocyte
[65] 3T3-L1 cells (2x10 cell/well) were cultured in DMEM containing 10% fetal bovine serum (FBS), 4.5 g/£ glucose, 2 mM glutamine, and antibiotic. After completely grown, the cells were cultured in DMEMFBS (prepared as described above) added with insulin (10 μg/m#), 0.25 μM dexamethasone, 0.5 mM isobutylmethylxanthine, 10 μM troglitazone (Sigma) which is an accelerator for adipocyte differentiation, and mineral water diluted with DMEM to hardnesses 100, 300, 500, 1000, 2000, 3000 and 4000, respectively, at 37 0C for 2 days. They were cultured further in media added with only insulin (10 μg/m#) at 37 0C for 3 days. Then, the media were replaced with DMEMFBS added with insulin, dexamethasone, isobutylmethylxanthine, and troglitazone every two days until the cells were used in the experiment. When 90 % or more of the cells showed an adipocyte phenotype by accumulation of lipid droplets, the cells were treated with mineral water diluted with DMEM to hardnesses 100, 300, 500, 1000, 2000, 3000 and 4000, respectively. After 48 hours at 37 0C, the lipid droplets in the cells were stained with 10% oil red O, extracted with ethanol, and quantified at 515 nm.
[66] The effect of the mineral water on inhibiting the adipocyte differentiation after the differentiation accelerator treatment was performed is shown in FIG. 4 according to the hardness. As shown in FIG. 4, after 5 days, the groups of a hardness 500 or higher showed 10 % or higher (approximately 10 % to approximately 40 %) inhibition against lipid generation.
[67]
[68] Experiment 5: Inhibitory Effect of Mineral Water on Expression of Genes Inducing
Adipocyte [69] While the differentiation of the preadipocyte to the adipocyte is induced by insulin, transcriptional expression of transcriptional factors such as PPARγ, C/EBP famil, ADD1/SREBP1 and the like increase. In this experiment, so as to investigate the effect of the mineral water on inhibiting expression of genes related to the adipocyte induction, the expression of the genes in a cell was analyzed using PPARγ through real time PCR.
[70]
[71] 1. Preparation of materials
[72] 3T3-L1 cells (6xlO5 cell / 25 cm2 T-flask) were cultured in DMEM containing 10% fetal bovine serum (FBS), 4.5 g/£ glucose, 2 mM glutamine, and antibiotic. After completely grown, the cells were cultured in DMEMFBS added with insulin (10 μg/ m#), 0.25 μM dexamethasone, 0.5 mM isobutylmethylxanthine, 10 μM troglitazone (Sigma) which is an accelerator for adipocyte differentiation, and mineral water diluted with DMEM to hardnesses 1000, 2000 and 4000, respectively, at 37 0C for 2 days. They were cultured further in media added with only mineral water and insulin (10 μg/ m-6) at 37 0C for 3 days. Then, the media were replaced with DMEMFBS added with insulin, dexamethasone, isobutylmethylxanthine, troglitazone, and mineral water every two days until the cells were used in the experiment. Total RNA were extracted when 90 % or more of the cells in a control group showed an adipocyte phenotype by accumulation of lipid droplets.
[73] The total RNA were extracted using Invisorb Spin Cell RNA Mini Kit (Invitek
GmbH, Germany). RNA were ψantified using Genensis 10 spectrophotometer (Thermo scientific, USA).
[74]
[75] 2. Analysis of gene expression using real time PCR
[76] Real time PCR was performed using SG Quantitect primer assay (Qiagen Inc, USA) for primers of a housekeeping gene (GAPDH) and a target gene (PPARγ), and using Sensimix onestep kit (Quantace, UK) with a template of from 1 pg to 100 ng of total RNA extracted from each cell. Then, an amplification curve and a lysis curve of each gene were analyzed to confirm specific amplification of the target sequence. mRNA expression level of each gene was quantified with a CT (threshold cycle) value, and standardized with a CT value of GAPDH.
[77] The effect of the mineral water on inhibiting the expression of the genes inducing adipocyte after treated with the accelerator for adipocyte differentiation is shown in FIG. 5. As shown in FIG. 5, as the hardness increases, the expression of PPARγ was inhibited, and the mineral water of a hardness 1000 showed 50 % inhibition against gene expression, the mineral water of a hardness 2000 showed 67 % inhibition against gene expression, and the mineral water of a hardness 4000 showed 95 % inhibition against gene expression, providing significant results.
[78]
[79] Experiment 6: Effect of Mineral Water on Weight Reduction and Lipid Control in
Fat Mouse Model
[80]
[81] In order to investigate the effect of the mineral water on weight reduction and lipid control in fat mouse model (db/db), animal experiments were performed using white rats, as follows.
[82]
[83] 1. Purchasing and breeding of experiment animals
[84] For the white rats, 3 weeks old male Spragμe-Dawley rats (Samtako, Korea) (weight:
80+10 g) were purchased. They were fed with cubed diets (Samyang Feed, Korea) and free water in an animal laboratory for 7 days (adaptation period). After the adaptation period, they were divided into 6 groups including a group fed with distilled water, and 5 groups fed with mineral water of hardnesses 100, 500, 1000, 2000, and 4000, respectively. The 6 groups of the white rats were fed with corresponding cubed diets and free water for 5 weeks (breeding period). During the breeding period, their weight variation and amount of water intake were measured. Thereafter, obesity inhibition experiments were performed on them. The animal room was maintained at a constant temperature and humidity condition (23+ 30C, 50+ 10% RH), and light was automatically controlled at 12 hours' intervals from 08: 00 to 20: 00.
[85]
[86] 2. Materials
[87] The mineral water of a hardness 4000 prepared in Preparation 2 was used without dilution, or after being diluted with distilled water to have hardnesses 100, 500, 1000 and 2000, respectively.
[88]
[89] 3. Measurement of body weight and body fat weight for each region
[90] Every white rat was measured for body weight at 2 o'clock every two days. The white rats were fed with weighted diets, and the remaining water was measured the next day to calculate the amount of daily water intake. At the end of the experiment, the rats were anesthetized. Then, the weights of the brown adipose fat (BAT) and the epididymal fat were measured before the perfusion, and the weights of the peritoneal fat and the visceral fat were measured after the perfusion, to compare the weights of the body fats for respective regions.
[91] The results are as follows.
[92]
[93] a) Variation of weight
[94] The distilled water group (the group of white rats fed with distilled water) and the mineral water groups (the groups of white rats fed with mineral water of hardnesses 100, 500, 1000, 2000 and 4000, respectively) were measured for variations of body weights according to the water intake for 3 weeks, and the results are shown in FIG. 6. As shown in FIG. 6, all of the mineral water groups showed significant effect of inhibiting body weight increase. The time "0" in FIG. 6 indicates the treatment time of the mineral water. From the comparison of the final body weights after the treatment, it was found that the mineral water groups had a significant inhibitory effect on the increase of the body weight, and 5 % to 25 % of the body weight was decreased for 3 weeks.
[95]
[96] b) Comparison of body fat weights of respective regions
[97] The variations of the body fat weights of respective regions according to the mineral water intake were compared and shown in FIG. 7. As shown in FIG. 7, the weights of the epididymal fats of the mineral water groups were not cμite different from the control group. For the mineral water group of a hardness 1000, the weight of the visceral fat was decreased by 20.2 % and the weight of the peritoneal fat was decreased by 19.4 % compared to the distilled water group.
[98]
[99] Experiment 7: Effect of Mineral Water on Weight Reduction and Lipid Control in
Fat Mouse Model
[100] Mineral water skin preparation was prepared using the mineral water according to the exemplary embodiment, and their compositions are listed in Table 2. In Table 2, the mineral water includes four groups of mineral waters having hardnesses 100, 1000, 2000 and 4000, respectively. For the four groups of mineral waters, the mineral water of a hardness 4000 prepared in Preparation 2 was used without dilution, or after being diluted to hardnesses 100, 1000 and 2000 with distilled water. For reference, the deep sea mineral water among the materials for the cosmetics may be referred to as 'sea water' regardless of the specifications. [101] Table 2 [Table 2] [Table ]
Figure imgf000014_0001
[102] Twenty grown women having local obesity or cellulite and having an age in the
2 range of 25 to 46 and a body mass index (BMI: weight (kg)/(heightxheight (m ))) in the range of 21 to 30 took part in the experiment. Each of the women was applied with the skin preparation prepared using the mineral water according to the hardness, on one thigh two times every day, i.e., in the every morning and in the every evening for 6 weeks. The effect was determined by the instrumental evaluation and the evaluation of the researcher before and after the application of the skin preparation for 8 weeks.
[103] The girth of the thigh was measured using a measuring tape (unit: cm) after marking a predetermined portion of the thighs. The measurement was conducted at two portions of the thigh, i.e., at upper end portion and a middle portion of the thigh. The values before and after the application were compared to each other using Student t test or Wilcoxon test as two-sided test, to analyze the statistical significance (significance level, α=0.05). Levene test and Kolmogorov-Smirnov test were performed to determine the uniformity of distribution and variance, and when it is determined to be uniform, the Student t test was used.
[104] Ultrasound-EuB 415 US scanner was used to measure the thicknesses of the subcutaneous fat and infiltrated cellulite through ultrasonic wave. The values measured before and after the application were compared to each other using Student t test or Wilcoxon test as two-sided test, to analyze the statistical significance (significance level, α=0.05). Levene test and Kolmogorov-Smirnov test were performed to determine the uniformity of distribution and variance, and when it is determined to be uniform, the Student t test was used.
[105] After 6 weeks' application of the skin preparation, the girth of the thigh was found to be 0.3 cm to 0.6 cm smaller in the inventive examples (applied with the skin preparation containing the mineral water) than in the comparative example (applied with the skin preparation containing the distilled water), providing a statistically significant result. However, the body weight of the subject did not varied during the experiment.
[106] FIG. 8 is a graph illustrating reduction rates of the subcutaneous fat and the cellulite after the application of the skin preparation containing the mineral water according to the exemplary embodiment. As shown in FIG. 8, the thigh applied with the skin preparation containing the mineral water had the subcutaneous fat reduced by 2.7 % compared to the state before the application (in the case of a hardness 100), providing a statistically significant result. In comparison, the comparative example did not show a significant variation.
[107] In addition, in comparison to the comparative example, the inventive examples, e.g., the case where the mineral water has a hardness of 1000 showed a cellulite reduction rate of approximately 15 %, providing a statistically significant result.
[108] From the above described results, it can be seen that the mineral water separated from the deep sea water is effective in subcutaneous fat reduction and cellulite reduction. Industrial Applicability
[109] Provided are mineral water containing a unique mineral, particularly, mineral water separated from deep sea water, and use thereof. The deep sea water composition is effective in inhibiting lipid generation with the help of the mineral components contained therein and thus can be applied to beverage and food compositions and skin cosmetics for anti-obesity and weight reduction.

Claims

Claims
[1] A mineral water for inhibiting differentiation or growth of fat cells, comprising a magnesium salt, a calcium salt, a potassium salt and a sodium salt, wherein a content ratio of magnesium (Mg):calcium (Ca):potassium (K):sodium (Na) in the mineral water is 3:0.5-1.5:0.5-1.5:0.5-1.5 by weight.
[2] The mineral water of claim 1, wherein a hardness of the mineral water is 100 to
4000 as calculated according to the following equation: Hardness = magnesium (mg/L) x 4 + calcium (mg/L) x 2.5.
[3] A mineral water for inhibiting differentiation or growth of fat cells, comprising a deep sea water or a solution of a magnesium salt, a calcium salt, a potassium salt and a sodium salt dissolved in a distilled water, wherein the magnesium salt, the calcium salt, the potassium salt and the sodium salt are separated from the deep sea water.
[4] The mineral water of claim 3, further comprising water such that a hardness of the mineral water is 100 to 4000 as calculated according to the following equation: Hardness = magnesium (mg/L) x 4 + calcium (mg/L) x 2.5.
[5] A composition for body fat reduction, body weight reduction or anti-obesity, comprising the mineral water of one of claims 1 to 4.
[6] A functional food for inhibiting differentiation or growth of fat cells, comprising the mineral water of one of claims 1 to 4.
[7] A skin external preparation for body weight reduction or fat cell removal, comprising the mineral water of one of claims 1 to 4.
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