KR20140013857A - Anti-obesity composition comprising mineral extract of deep sea water - Google Patents

Abstract

The present invention relates to a novel use of a mineral extract of deep sea water which provides a composition with an anti-obesity function by increasing the decomposition of neutral fat and controlling the lipid metabolism and the differentiation of fat cells. The mineral extract of deep sea water contains Mg and Ca in a composition ratio of 3:1.

Description

Anti-obesity Composition Comprising Mineral Extract of Deep Sea Water}

The present invention relates to an anti-obesity functional composition containing the deep sea water mineral extract as an active ingredient.

Modern society has a very serious economic and social burden due to various complications and rapidly increasing prevalence caused by obesity. Obesity is known to be closely associated with the development of diabetes, hypertension, dyslipidemia, osteoarthritis, cardiovascular disease, and some cancers.

To date, prevention and treatment of obesity have been applied to clinical trials such as non-pharmaceutical therapies such as diet and exercise, or a variety of anti-obesity drugs that directly control metabolic processes directly involved in obesity. However, in the case of clinical application of anti-obesity drugs, most of them are not used in clinic due to serious side effects and only a few anti-obesity drugs are used carefully. Therefore, the necessity of a new anti-obesity prevention and treatment is constantly being demanded, and researches that find the solution in natural resources are actively conducted.

Deep sea water (DSW), one of natural resources, is known to be rich in minerals such as Mg, Ca, and K. In particular, Mg and Ca have been reported to act as antidiabetic substances. Korean Unexamined Patent Publication No. 10-2011-0110587 discloses a pharmaceutical composition for improving and preventing diabetes containing a mineral composition as an active ingredient.

However, until now, no anti-obesity effect has been suggested by various depth-depth mineral extracts of Mg and Ca by 3: 1.

Accordingly, it is an object of the present invention to provide a functional composition comprising the deep sea water mineral extract having an anti-obesity effect as an active ingredient.

The above object of the present invention comprises the step of preparing a deep sea water mineral extract for each hardness consisting of Mg and Ca ratio of 3: 1; Cytotoxicity verification step by the deep sea water mineral extracts by hardness; Adipocyte differentiation and fat accumulation using Oil Red O staining; Investigation of the expression of fat cell differentiation-related genes by deep sea water mineral extracts by hardness; A step of investigating gene expression related to lipid metabolism by the deep seawater mineral extract by hardness; Obesity-related adipokines expression was achieved through the step of investigation.

The present invention has an excellent effect of providing an anti-obesity functional composition using the deep sea water mineral extracts.

Figure 1 is a graph showing the cell survival rate of the deep seawater mineral extract by hardness in 3T3-L1 adipocytes.
Figure 2 is a graph showing the cell differentiation and fat accumulation of 3T3-L1 adipocytes by the deep seawater mineral extracts by hardness.
Figure 3 is a graph showing the mRNA expression level of 3T3-L1 adipocyte differentiation-related genes by the deep seawater mineral extracts by hardness.
Figure 4A is a graph showing the mRNA expression level of lipogenic genes (lipogenic genes) by marine deep water mineral extracts by hardness in 3T3-L1 adipocytes.
Figure 4B is a graph showing the mRNA expression of lipolytic genes (lipolytic genes) by the deep seawater mineral extracts by hardness in 3T3-L1 adipocytes.
5 is a result showing the degree of adipokines expression by the deep seawater mineral extract by hardness in 3T3-L1 adipocytes.

Hereinafter, specific examples of the present invention will be described in detail with reference to Examples and Experimental Examples, but the scope of the present invention is not limited only to these examples.

Example 1 Preparation of Deep Sea Water Mineral Extracts by Hardness

Deep sea water (DSW) was collected at a depth of 0.5 km, 6.7 km from Goho-gun, Ohori (Gangwon-do, South Korea), to remove phytoplankton and marine microorganisms using a microfiltration system. The filtered deep seawater was passed through a reverse osmosis membrane to obtain brine and demineralized water. The balanced deep seawater was prepared by mixing the brine and demineralized water so that the composition ratio of the mineral Mg: Ca is 3: 1, and then diluted it sequentially according to hardness to use the deep sea water mineral extract of the present invention.

In general, hardness is defined as the property of water that represents the total concentration of hydrogen ions expressed as polyvalent ions such as calcium, magnesium, iron, manganese, strontium, zinc and calcium carbonate. In the present invention, the hardness of the deep sea water mineral extract was calculated by the following Equation 1 based on the concentration of calcium and magnesium ions.

Table 1 below shows the composition of the mineral components of the deep sea water and the balanced deep sea water with a hardness of 4,000. All of the deep sea water mineral extracts used in the present invention were filtered with a 0.2 μm bottle-top filter (Fisher Scientific Inc, IL, USA) before the experiment.

Mineral Original DSW (mg / kg) Balanced DSW (mg / kg) Ca 417.2 314 Mg 1,299.8 950 K 388.2 2.1 Na 10,794.0 250 Cl 18,607.0 665 SO ₄ 2,624.0 132

Experimental Example 1: Validation of Cytotoxicity of Marine Deep Water Mineral Extracts

In order to investigate the cytotoxicity of the deep seawater mineral extract obtained in Example 1, MTT analysis was performed using fat precursor cells 3T3-L1 (Korea Cell Line Bank, Seoul, Korea; KCLB No. 10092.1). More specifically, Preconfluent 3T3-L1 cells were cultured in 96-well plates at 5 × 10 ⁴ cells / well for 24 hours. After removing the culture medium, it was incubated for 24 hours and 48 hours by replacing with DMEM (Hyclone Laboratories, Inc., UT, USA) medium dissolved in deep sea water mineral extract consisting of different hardness, that is, hardness range of 500 to 4000. 100 uL of MTT (3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide, Sigma Aldrich, St. Louis, MO, USA) solution (5 mg / mL) was added to the cultured cells. After reacting for 4 hours, the supernatant was removed and 100 uL DMSO was added. Cell viability was calculated by measuring absorbance at 490 nm (VersaMax microplate reader; Sunnyvale, CA, USA).

Experimental results As shown in Figure 1, the hardness value of the investigated deep seawater mineral extracts did not show significant cytotoxicity in the fat precursor cells 3T3-L1 all in the range of 500 to 4000.

Example 2 Cell Culture and Adipocyte Differentiation

3T3-L1 cells were cultured in DMEM medium containing 10% calf serum (Hyclone Laboratories, Inc., UT, USA) and antibiotic penicillin-streptomycin (Hyclone Laboratories, Inc., UT, USA). To induce cell differentiation, post-confluent 3T3-L1 cells were harvested on day 2 in MDI induction medium, 10% fetal bovine serum (Hyclone Laboratories, Inc., UT, USA), 0.5 mM 3-isobutyl-1-methylxanthine (IBMX, In DMEM medium consisting of Sigma Aldrich, St. Louis, MO, USA), 1 mM dexamethasone (Sigma Aldrich, St. Louis, MO, USA) and 10 μg / mL insulin (Sigma Aldrich, St. Louis, MO, USA) Incubated for 2 days. The medium was then replaced with medium containing 10 μg / mL insulin.

Experimental Example 2: Adipocyte Differentiation and Fat Accumulation by Oil Red O Staining

In order to observe the inhibition of adipocyte differentiation by the deep seawater mineral extract by hardness, lipid droplet formation of the differentiated adipocytes in the medium dissolved in the deep seawater mineral extract was observed using Oil Red O staining. That is, 3T3-L1 adipocytes differentiated for 8 days were washed with PBS, fixed with 10% formalin at room temperature for 1 hour, washed with 60% isopropanol, and then completely dried. The fixed cells were stained with 0.1% Oil red O solution dissolved in a working solution (isopropanol: water = 3: 2) at room temperature for 1 hour and then washed with distilled water. The degree of adipocyte differentiation was observed using a light microscope (Nikon eclipse TS 100, NIS-Elements Imaging Software ver. 4.0, Nikon, Tokyo, Japan). The stained oil droplets were dissolved with isopropanol, and then measured by a spectrophotometer (VersaMax microplate reader; Sunnyvale, CA, USA) at 590 nm to observe the degree of fat accumulation.

In addition, triglyceride content, a major feature of adipocyte differentiation, was investigated using the EnzyChrom ^™ Triglyceride assay Kit (Bioassay Systems, Hayward, CA, USA). That is, 3T3-L1 cells differentiated from deep seawater mineral extracts by hardness were washed with PBS, harvested with homogenizing solution (50 mM Tris, 154 mM KCl, 1 mM EDTA, pH 7.4), and then sonicated. Dissolved. In order to remove the fat layer was centrifuged for 5 minutes at 3,000 rpm, 4 ℃ and the obtained supernatant was measured triglyceride and protein content.

As shown in FIG. 2, during the differentiation period, adipocyte differentiation (FIG. 2A) and fat accumulation (FIG. 2B) were suppressed hardness-dependently in the hardness value range of 500 to 2000 of the deep seawater mineral extract. In addition, the hardness-dependent significant decrease was observed in the triglyceride content of 3T3-L1 cells with respect to the hardness value of the deep sea water mineral extract (FIG. 2C). In addition, T0070907 (Cayman chemical company, Ann Arbor, Michigan, USA) showed an effect of inhibiting the differentiation of fat cells as a PPARγ antagonist. Therefore, inhibition of adipocyte differentiation and fat accumulation was not judged to be due to the toxicity of deep sea mineral extract.

Experimental Example 3: Expression of fat cell differentiation related genes by deep seawater mineral extract

In order to elucidate the inhibitory mechanism by deep seawater mineral extract during adipocyte differentiation, the expression level of important transcription factors such as PPARγ and C / EBPα and glucose transporter Glut4, which are essential components in adipocyte differentiation, through quantitative real-time PCR (polymerase chain reaction) Respectively.

In other words, Total RNA was isolated from 3T3-L1 cells on day 8 of differentiation using Trizol (Invitrogen Life Technologies, Carlsbad, Calif., USA). cDNA was synthesized using PrimeScript ^™ 1 ^st strand cDNA synthesis kit (Takara Bio INC., Shiga, Japan) as a template using the isolated total RNA according to the manufacturer's manual. Real-time PCR was performed by using the FastStart SYBR Green Master (Roche Diagnostics, Mannheim, Germany) to react the synthesized cDNA with the primers shown in Table 2 below, followed by ABI Prism 7300 Sequence Detection System (Applied Biosystems, Foster City, Three times).

The gene expression degree of the present invention was normalized to the Ct value of the endogenous reference gene actin using the delta cycle threshold (Ct) value obtained from the real-time PCR.

As shown in FIG. 3, the deep sea water mineral extract significantly inhibited the expression of PPARγ, C / EBPa and Glut4 depending on hardness for hardness values ranging from 500 to 2000. Therefore, the deep sea water mineral extract was determined to be involved in the regulation of adipocyte differentiation of adipocytes 3T3-L1.

Experimental Example 4: Investigation of the expression of liposynthesis and lipolysis gene by deep sea water mineral extract

To observe the regulation of adipocyte-specific genes by deep seawater mineral extracts, lipogenic genes such as sterol regulatory element-binding protein 1c (SREBP1c) and fatty acid synthase (FAS) and lipolytic genes (lipolytic) gene), namely, the expression levels of adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL) and perilipin (PLIN) were examined by the method shown in Experimental Example 3.

Experimental results As shown in Figure 4, deep sea water mineral extract significantly inhibited the expression of liposynthesis (FIG. 4A) and lipolysis (FIG. 4B) genes hardness-dependently for hardness values in the range of 500 to 2000. Therefore, the deep sea water mineral extract was determined to regulate fat metabolism including fat synthesis and lipolysis.

Experimental Example 5 Control of Obesity-Related Adipokines Expression by Deep Sea Water Mineral Extracts

In order to observe the adipokines gene expression by the deep sea water mineral extract, real-time PCR and western blot analysis shown in Experiment 3 was performed.

The western blot analysis was performed by washing cold 3BS 3T3-L1 cells cultured in the deep seawater mineral extract by hardness, then harvested RIPA buffer (50 mM NaCl, 10 mM Tris, 0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate, 5 mM EDTA and 1 mM Na ₃ VO ₄ , pH 7.4). Total cell protein (40 ㎍) was separated by SDS-PAGE, and then transferred to the nitrocellulose membrane (Whatman, Dassel, Germany), the membrane was blocked for 1 hour in 5% skim milk. Subsequently, after overnight reaction at 4 ° C. with a primary antibody (1: 1000), the membrane was washed with Tris-buffered saline (TBS) containing 0.1% Tween-20, and then a secondary antibody (horseradish peroxidase conjugated antibodies). , 1: 3000) for 1 hour at room temperature. The antibody-bound membrane was reacted with an enhanced chemiluminescence (ECL) solution (Amersham Bioscience, Buckinghamshire, UK) and confirmed by exposure to an X-ray film. The primary antibodies used in the present experiment were anti-adiponectin (Cell Signaling Technology, Danvers, MA, USA), anti-leptin and anti-β-actin (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), 2 The primary antibodies were anti-mouse IgG-HRP and anti-rabbit IgG-HRP (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA).

As shown in FIG. 5, the deep seawater mineral extract significantly reduced adiponectin mRNA expression and mildly increased leptin mRNA expression depending on hardness. In contrast, deep seawater mineral extracts significantly increased leptin protein expression. Moreover, T0070907, dissolved in artificial mineral water with a magnesium and calcium ratio of 3: 1, was a PPARγ antagonist and significantly increased leptin expression.

Through the above experimental results, deep sea water mineral extract was determined to be a novel natural substance having an anti-obesity effect by increasing adipocyte differentiation, inhibiting lipid metabolism and triglyceride degradation.

As described above, the present invention has an excellent effect of providing an anti-obesity functional composition containing the deep sea water mineral extract as an active ingredient, so it is a very useful invention in the food and pharmaceutical material industry.

Claims (4)

Anti-obesity functional composition containing a deep sea water mineral extract as an active ingredient.
The anti-obesity composition according to claim 1, wherein the deep sea water mineral extract has a hardness value in the range of 500 to 2000 calculated by Equation 1 below:
[Equation 1]

The anti-obesity composition according to claim 1 or 2, wherein the deep sea water mineral extract has a Mg: Ca ratio of 3: 1.
The composition of any one of claims 1 to 3, wherein the composition is an anti-obesity agent.

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