WO2014017689A1

WO2014017689A1 - Anti-obesity composition comprising mineral extract of deep sea water

Info

Publication number: WO2014017689A1
Application number: PCT/KR2012/006185
Authority: WO
Inventors: Yun Hee Shon; Byung Geun HA; Deok-Soo MOON
Original assignee: Kyungpook National University Hospital
Priority date: 2012-07-27
Filing date: 2012-08-03
Publication date: 2014-01-30
Also published as: KR20140013857A

Abstract

The present invention relates to a novel application of mineral extract of deep sea water having inhibition of adipocyte differentiation and lipid metabolism and decrease of triglyceride content, which has an effect on providing anti-obesity composition.

Description

ANTI-OBESITY COMPOSITION COMPRISING MINERAL EXTRACT OF DEEP SEA WATER

The present invention relates to anti-obesity composition comprising mineral extract of deep sea water.

Today we are faced with very serious economic and social burdens due to a variety of complications and rapidly increasing prevalence caused by obesity. Obesity is known to have a close relationship with diseases such as diabetes, hypertension, dyslipidemia, osteoarthritis, cardiovascular disease and cancer.

Until now, nonpharmacologic therapies such as diet and exercise, or many types of anti-obesity drugs that regulate metabolic processes directly involved in obesity have been applied in clinic for prevention and treatment of obesity. However, anti-obesity drugs have not been widely used in most clinical application due to serious side effects and only a few anti-obesity drugs have been used carefully. The need thus, for new method of obesity prevention and treatment has been constantly required and natural resources are paying very much attention to find a solution.

Deep sea water (DSW), one of natural resources, is known to contain high content of minerals including magnesium (Mg), calcium (Ca) and potassium (K), especially Mg and Ca have been already reported as effective components on diabetes. Korean Patent Publication No. 10-2011-0110587 discloses pharmaceutical compositions containing minerals as effective components for diabetes improvement and prevention.

However, there has been no report up to date on mineral extract of DSW with different hardness consisting essentially of mineral ratio of Mg:Ca = 3:1.

Accordingly, the object of the present invention is to provide anti-obesity composition comprising mineral extract of deep sea water.

The above object was achieved as follows:

preparing mineral extract of DSW with different hardness consisting essentially of mineral ratio of Mg:Ca = 3:1;

testing cytotoxicity of the above mineral extract of DSW;

testing adipocyte differentiation and lipid accumulation by Oil Red O staining; and

testing expression of adipogenic genes including adipokines.

The present invention has an effect on providing the anti-obesity composition from the mineral extract of deep sea water.

Figure 1 is a graph showing an effect of mineral extract of DSW on cell viability in 3T3-L1 preadipocytes.

Figure 2 is showing an effect of mineral extract of DSW on adipocyte differentiation and lipid accumulation in 3T3-L1 preadipocytes.

Figure 3 is a graph showing an effect of mineral extract of DSW on mRNA expression of adipogenic genes in 3T3-L1 adipocytes.

Figure 4a is a graph showing an effect of mineral extract of DSW on mRNA expression of lipogenic genes in 3T3-L1 adipocytes.

Figure 4b is a graph showing an effect of mineral extract of DSW on mRNA expression of lipolytic genes in 3T3-L1 adipocytes.

Figure 5 is showing an effect of mineral extract of DSW on expression of adipokines in 3T3-L1 adipocytes.

The present invention hereinafter, will be described by the following examples in more detail. However, such examples are only to illustrate the invention and they do not restrict the present invention.

Example 1: Preparation of mineral extract of deep sea water

Deep sea water (DSW), pumped up from a depth of 0.5 km and a distance of 6.7 km off Oho-Ri, Goseong (Gangwon-Do, Korea), was filtered by microfilter system to remove the phytoplankton and marine micro-organisms. The filtered DSW was passed through the reverse osmotic membrane and the brine and desalinated water were obtained. The above brine and desalinated water were mixed to prepare the balanced DSW with mineral ratio of Mg:Ca = 3:1. The balanced DSW was serially diluted by hardness and used in the present invention.

Generally, the hardness is defined as the characteristic of water that represents the total concentration of polyvalent ions such as calcium, magnesium, iron, manganese, strontium, zinc and hydrogen ions expressed as calcium carbonate. In this　present invention, we defined the hardness of mineral extract of DSW focusing on the concentration of calcium and magnesium ions. The hardness values were calculated according to the following Math Figure 1:

MathFigure 1

The following Table 1 shows mineral contents of original and balanced DSW of hardness 4,000. All mineral extract of DSW used in the present invention was sterilized by a 0.2 ㎛ bottle-top filter (Fisher Scientific Inc, IL, USA ) before experiments.

Table 1

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
SO₄	2,624.0	132

Example 2: Cytotoxicity test of mineral extract of DSW

To determine the effect of mineral extract of DSW obtained from the above example 1 on cytotoxicity, MTT assay was performed using 3T3-L1 preadipocyte cells (Korean Cell Line Bank, Seoul, Korea; KCLB No. 10092.1). More particularly, preconfluent 3T3-L1 preadipocytes　were seeded in 96-well plates at a density of 5 × 10⁴cells/well. At 24 h after plating, all media were removed from the wells and then changed with DMEM (Dulbecco’s modified Eagle’s medium, Hyclone Laboratories, Inc., UT, USA) dissolved in each mineral extract of DSW with different hardness values within the range of 500 to 4,000. After incubation for 24 h and 48 h, 100 μL of　MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma Aldrich, St. Louis, MO, USA) solution (5 ㎎/mL) were added followed by 4 h. Thereafter,　the supernatant was removed and 100 μL DMSO were added. The cell viability was calculated by reading the absorbance at 490 ㎚ (VersaMax microplate reader; Sunnyvale, CA, USA).

As shown in Figure 1, all the different hardness values 500 to 4,000 of mineral extract of DSW examined had no significant cytotoxicity against 3T3-L1 preadipocytes.

Example 3: Cell culture and adipocyte differentiation

3T3-L1 cells were maintained in DMEM containing 10% calf serum (Hyclone Laboratories, Inc., UT, USA) and antibiotics (penicillin-streptomycin, Hyclone Laboratories, Inc., UT, USA)). To induce differentiation, 2-day post confluent 3T3-L1 cells were incubated in MDI induction medium including DMEM containing 10% fetal bovine serum (Hyclone Laboratories, Inc., UT, USA), 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 1 mM dexamethasone and 10 ㎍/mL insulin (Sigma Aldrich, St. Louis, MO, USA) for 2 days. After that, the medium was changed to medium containing 10 ㎍/mL insulin.

Example 4: Test of adipocyte differentiation and lipid accumulation by Oil Red O staining

To determine whether mineral extract of DSW inhibits adipocyte differentiation, Oil-Red O staining was carried out to visualize the formation of lipid droplet in differentiated adipocytes under medium containing different hardness of mineral extract of DSW. In brief, after differentiation for 8 days, 3T3-L1 adipocytes were washed with PBS, fixed with 10% formalin for 1 h at room temperature, washed with 60% isopropanol and dried completely. The above fixed cells were then stained with a 0.1% Oil red O in working solution that made up isopropanol diluted (3:2) in distilled water for 1 h at room temperature and then washed with distilled water. Image acquisition was performed using a light microscope (Nikon eclipse TS 100, NIS-Elements Imaging Software ver. 4.0, Nikon, Tokyo, Japan). Stained oil droplets were dissolved with isopropanol and quantified by spectrophotometry at 590 ㎚ (VersaMax microplate reader; Sunnyvale, CA, USA).

In addition, the content of triglyceride, a major characteristic of adipocyte differentiation, was measured using a commercial EnzyChrom™ Triglyceride assay Kit (Bioassay Systems, Hayward, CA, USA). Briefly, 3T3-L1 cells differentiated under different hardness of mineral extract of DSW were washed with PBS, collected in homogenizing solution (50 mM Tris, 154 mM KCl, 1 mM EDTA, pH 7.4) and sonicated to homogenize the cell suspension. The residual cell lysate was centrifuged at 3,000 rpm for 5 min at 4℃ to remove the fat layer. The supernatants were assayed for triglyceride content and protein content.

As shown in Figure 2, during differentiation period, the mineral extract of DSW inhibited adipocyte differentiation (Figure 2A) and lipid accumulation (Figure 2B) in a hardness-dependent manner within the range of 500 to 2,000. The mineral extract of DSW significantly decreased triglyceride content in a hardness-dependent manner as well (Figure 2C). The treatment of T0070907 (Cayman chemical company, Ann Arbor, Michigan, USA), a synthetic PPARγ antagonist, also exhibited inhibitory effects of adipocyte differentiation. Therefore, the decrease in adipocyte differentiation and lipid accumulation was considered to be not due to toxic effect by the mineral extract of DSW.

Example 5: Test for expression of adipogenic genes by the mineral extract of DSW

To investigate the inhibitory mechanism of the mineral extract of DSW during the adipocyte differentiation, the expression levels of PPARγ and C/EBPα, which are key transcriptional factors, and Glut4, which is a glucose transporter, essential components for terminal adipocyte differentiation, were measured by quantitative real-time PCR (polymerase chain reaction).

In brief, total RNA was isolated from differentiated 3T3-L1 cells at day 8 using Trizol (Invitrogen Life Technologies, Carlsbad, CA, USA). cDNA was synthesized using a PrimeScriptTM 1^st strand cDNA synthesis kit (Takara Bio INC., Shiga, Japan) according to the manufacturer's instructions. Real-time PCR was performed with FastStart SYBR Green Master (Roche Diagnostics, Mannheim, Germany) in an ABI Prism 7300 Sequence Detection System (Applied Biosystems, Foster City, CA) in triplicate. The primer sequences are listed in Table 2 below.

Table 2

The relative expression levels of the target genes to the expression level of the endogenous reference gene actin were calculated using the delta cycle threshold (Ct) method.

As shown in Figure 3, the mineral extract of DSW intensively inhibited expressions of adipogenic genes including PPARγ, C/EBPα, and Glut4 in a hardness-dependent manner within the range of 500 to 2,000. Therefore, the mineral extract of DSW was considered to affect the regulatory mechanism for adipocyte differentiation in 3T3-L1 preadipocytes.

Example 6: Test for expression of lipogenic and lipolytic genes by the mineral extract of DSW

To further investigate whether the mineral extract of DSW regulates adipogenic target genes such as sterol regulatory element-binding protein 1c (SREBP1c) and fatty acid synthase (FAS) for lipogenesis and adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL) and perilipin (PLIN) for lipolysis, quantitative RT-PCR analysis was performed in the same manner as shown in Example 5.

As a result, the mineral extract of DSW intensively inhibited expressions of both lipogenic (Figure 4a) and lipolytic (Figure 4b) genes in a hardness-dependent manner within the range of 500 to 2,000. Thus, the mineral extract of DSW was found to regulate adipocyte functions including lipogenesis and lipolysis.

Example 7: Test for expression of adipokines regulated to obesity

To determine the effect of the mineral extract of DSW on adipokines gene expression, quantitative RT-PCR and Western blot analysis were performed. The RT-PCR was performed in the same manner as shown in Example 5. For Western blot analysis, cells were washed with ice-cold PBS, scraped on ice and lysed in RIPA buffer (50 mM NaCl, 10 mM Tris, 0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate, 5 mM EDTA and 1mM Na₃VO₄, pH 7.4). Total cell proteins (40 ㎍) were separated by SDS-PAGE and transferred to a nitrocellulose membrane (Whatman, Dassel, Germany). The membrane was blocked with 5% skim milk for 1 h and incubated with primary antibodies (diluted 1:1,000) overnight at 4℃. After washing with Tris-buffered saline (TBS) containing 0.1% Tween-20, the membrane was then incubated with horseradish peroxidase conjugated secondary antibodies (diluted 1:3,000) for 1 h at room temperature. Antibody binding on the nitrocellulose membrane was detected with an enhanced chemiluminescence (ECL) solution (Amersham Bioscience, Buckinghamshire, UK) and X-ray film. The primary antibodies used in the present invention were anti-adiponectin (Cell Signaling Technology, Danvers, MA, USA), anti-leptin and anti-β-actin (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and the secondary antibodies were anti-mouse IgG-HRP and anti-rabbit IgG-HRP (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA).

As shown in Figure 5, the mineral extract of DSW decreased expression of adiponectin and a little increased expression of leptin in a hardness-dependent manner. However, in case of leptin expression, Western blot analysis revealed that the mineral extract of DSW strongly increased expression of leptin. Moreover, it was also found that T0070907, a PPARγ antagonist and artificial mineral water (Mg:Ca = 3:1) containing magnesium and calcium, intensively increased leptin expression.

Taken together, the mineral extract of DSW was found to have anti-obesity effect including the inhibition of adipocyte differentiation, lipogenesis and lipolysis as well as the increase in leptin expression, an anti-obesity adipokine.

As described above, the present invention has an effect on providing anti-obesity composition comprising mineral extract of DSW, so that is a very useful invention in food and drug material industry.

Claims

An anti-obesity composition comprising mineral extract of deep sea water.
The anti-obesity composition according to claim 1, wherein the mineral extract of deep sea water has the hardness values within the range of 500 to 2,000 calculated by Math Figure 1 below:

[Math Figure 1]
The anti-obesity composition according to claim 1 or 2, wherein the mineral extract of deep sea water has mineral ratio of Mg:Ca = 3:1.
The anti-obesity composition as in one of claims 1 to 3, wherein the composition is an anti-obesity drug.