KR101515842B1 - Pharmaceutical composition for prevention and treatment of hyperglycemia and type 2 diabetes comprising mineral extract of deep sea water - Google Patents

Abstract

The present invention relates to a composition for prevention and treatment of hyperglycemia and obesity type 2 diabetes mellitus containing a deep sea water mineral extract having a weight ratio of Mg: Ca of 3: 1 and a hardness of 1,000 to 2,000 ppm as an active ingredient, Inventing deep sea water (DSW); Comparing the fasting glucose and glucose load to the control group after administration of the deep sea water obtained above to HFD-induced obesity rats; Examining gene expression associated with glucose synthesis, glycogen synthesis, glycogen synthesis and degradation, sugar oxidation; The present invention has been accomplished by investigating glucose uptake in adipocytes and examining the phosphorylation of AMPK and glucose uptake related signal molecules. The present invention has an excellent effect of inhibiting increase of blood glucose and restoring lowered glucose tolerance.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for prevention and treatment of hyperglycemia and obesity type 2 diabetes, which comprises a mineral extract of deep sea water as an active ingredient. The pharmaceutical composition for prevention and treatment of hyperglycemia and type 2 diabetes comprises:

The present invention relates to a composition for prevention and treatment of hyperglycemia and obesity type 2 diabetes mellitus containing deep sea water mineral extract as an active ingredient.

It is known that the main cause of Type 2 diabetes, which is a serious health threat worldwide, is high-fat diet, mainly saturated fatty acid-rich meals. People with type 2 diabetes are resistant to insulin, a typical obesity that removes sugar from the blood into muscle or fat tissue and stores it as an energy source. In addition, since normal glucose uptake does not proceed, the ability of glucose tolerance is lowered, resulting in hyperglycemia due to persistent glucose in the blood.

Glucose homeostasis is regulated through complex metabolic processes such as hormone transfer processes between liver, fat, muscle, pancreas, and brain tissue. Especially in the liver, glucose levels are controlled through metabolic processes such as gluconeogenesis, glycogenesis, glycogenolysis, and glucose oxidation. Glycogen synthase (GS) and liver glycogen phosphorylase (LGP) are involved in glycogen metabolism. Glucokinase (GK), a glucose oxidase, is known to be involved in glycogen metabolism, and phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase) And citrate synthase (CS) enzymes.

Glucose uptake occurs in the terminal tissues that are responsible for most of the metabolism, such as energy storage or consumption, in the body, such as muscle or adipose tissue, and it is known that various signaling systems act. The major glucose uptake-related signaling pathway is the insulin signaling pathway and the 5'AMP-activated protein kinase (AMPK) signaling pathway. Finally, glucose transporter 4 (GLUT4), which directly injects external sugar into the cell by the signal transmission, is increased (cell membrane movement), thereby increasing glucose uptake. The insulin signal transduction system is present in all tissues, but specifically acts on the muscle and adipose tissue to bind the insulin receptor present in the cell membrane, and then the insulin receptor substrate 1 (IRS-1) binds to the receptor Sugar absorption occurs through the activity of successive lower signaling substances. The AMPK signaling system is primarily responsible for glucose uptake in muscle tissue in the state of energy deficiency, such as muscle contraction and malnutrition. In other tissues, the pancreas is involved in insulin secretion, and the liver is involved in glucose synthesis and lipid metabolism inhibition.

Korean Patent No. 10-0911949 discloses an anti-obesity use of deep-sea mineral water having a hardness of 100 to 4000 and containing Mg: Ca: K: Na = 3: 0.5-1.5: 0.5-1.5: 0.5-1.5 by weight. And Patent Document 10-2010-76512 discloses rice cooked in a range of hardness of 100 to 4,000 mg / L using mineral demineralized water derived from deep seawater of the sea, and rice prepared by using the same. However, in the case of hyperglycemic or obesity type 2 The mixing ratio and hardness of Mg: Ca most preferable for prevention and treatment of diabetes are not implied.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for prevention and treatment of hyperglycemia and obesity type 2 diabetes mellitus containing deep sea water mineral extract as an active ingredient.

Another object of the present invention is to provide a health functional food for prevention and treatment of hyperglycemia and obesity type 2 diabetes mellitus containing deep sea water mineral extract as an active ingredient.

The above object of the present invention is achieved by a method for producing a deep sea water (DSW) having a Mg: Ca weight ratio of 3: 1 and a hardness of 1,000 to 2,000 ppm; Comparing the fasting glucose and glucose load to the control group after administration of the deep sea water obtained above to HFD-induced obesity rats; Examining gene expression associated with glucose synthesis, glycogen synthesis, glycogen synthesis and degradation, sugar oxidation; Investigating glucose uptake in adipocytes and examining phosphorylation of AMPK and glucose uptake related signaling molecules.

The present invention provides a pharmaceutical composition for preventing and treating anti-obesity and fatty liver, which comprises an extract of deep sea water as an active ingredient. The deep sea water mineral extract preferably comprises 0.01 to 99% by weight based on the total weight of the pharmaceutical composition.

The compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions.

The composition of the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions, Examples of carriers, excipients and diluents that can be included in the composition containing the extract include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like. These solid preparations are prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose, lactose, do. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Examples of liquid formulations for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used diluents, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like.

In addition, the present invention provides a health functional food for preventing and treating an anti-obesity and fatty liver containing a deep sea water mineral extract as an active ingredient. The deep sea water mineral extract preferably comprises 0.01 to 99% by weight based on the total weight of the health functional food composition.

The weight ratio of Mg: Ca in the deep sea mineral extract of the present invention was found to be most preferably 3: 1 and most preferably 1,000 to 2,000 ppm. Experimental results were limited to this, and the effect was insignificant when hardness was below 500 and there was no difference in efficacy above 2,000.

The composition comprising deep sea water of the present invention can be variously used for food and beverage for anti-obesity and fatty liver prevention and treatment. Examples of the food to which the deep sea water of the present invention can be added include various foods, beverages, gums, tea, vitamin complexes, health supplement foods and the like, and they can be used in the form of powders, granules, tablets, capsules or drinks.

INDUSTRIAL APPLICABILITY The present invention has an excellent effect of suppressing an increase in blood glucose and restoring a reduced glucose tolerance.

FIG. 1 is a graph showing the effects of the deep sea water mineral extract of the present invention (A) on the fasting blood glucose level and (B) glucose consumption.
FIG. 2 is a graph showing the effects of the deep sea water mineral extract of the present invention on gene expression related to (A) glucose synthesis, (B) glycogen synthesis and degradation, and (C) glucose oxidation.
FIG. 3 is a graph showing the effect of the deep sea water mineral extract of the present invention on 2-NBDG uptake of 3T3-L1 adipocytes.
FIG. 4 is a photograph showing the effects of the deep sea water mineral extract of the present invention on (A) AMPK, ACC1 phosphorylation, (B) IRS1 phosphorylation, and GLUT4 expression.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.

Example : Inventive Balance Deep sea water ( balanced DSW ) Produce

The original deep sea water (DSW), which was pulled up from a depth of 0.5 km at a distance of 6.7 km from the Auri side of Goseong-gun, was filtered using a microfilter system (Sinopex, Pohang, Korea). The filtered DSW was passed through a reverse osmosis membrane to obtain brine and desalted water. The balanced DSW of the present invention having a weight ratio of magnesium and calcium of 3: 1 was prepared by mixing brine and desalted water, and the balanced DSW was continuously diluted according to the hardness. The inventors defined the hardness of the inventive DSW with an emphasis on the concentration of magnesium and calcium and the hardness was calculated according to the following formula.

Table 1 shows the DSW sample mineral content of the original and inventive hardness of 4,000 ppm. All balanced DSWs used in the experiments were sterilized by passage through a 0.2 μm Bartlett Top filter (Fisher Scientific Inc., IL, USA).

Experimental Example  1: invention Deep sea water  Effects on Fasting Blood Sugar and Glucose Loading

All animal experiments were conducted in accordance with the guidelines of the Animal Ethics Committee of Kyungpook National University and the experimental protocol was approved by the above-mentioned committee (Approval No. KNU 2012-88). C57BL / 6J rats were used as animal models in obese and diabetic rats to elucidate the effects of the present invention on body weight and fasting blood glucose. All experiments were performed on 26 week old male rats and the rats were housed in air-conditioned room at 22 ° C ± 2 ° C and relative humidity 40% ± 5%, 8:00 to 20:00. All rats were fed a pellet diet (LabDiet 5L79; Charles River, Wilmington, Mass., USA). After 1 week of pre-feeding, 70 C57BL / 6J mice (6 weeks old) And divided into five groups. Each group of five C57BL / 6J mice received a control diet (D12450B; Research Diets, normal diet) or high-fat diets (HFD; D12451; Research Diets) or DSW (500, 1,000 or 2,000 ppm) Were taken with HFD. Body weight, drinking water and food intake were measured every 3 days and rats were fasted for 18 hours at the end of feeding. Blood was collected from the heart by blood collection under anesthesia with zolethyl (Virbac SA, Carros, France) and stored at -70 ° C until used in the experiment.

To determine fasting blood glucose, blood was collected from the tail vein 4 hours after the rat diet was restricted. Blood (10 μL) was added to distilled water (30 μL) and 20% (w / v) trichloroacetic acid aqueous solution (40 μL) was added to the test tube and stored on ice. The mixture was centrifuged at 12,000 × g for 10 min at 4 ° C. and the supernatant (10 μL) was used to identify the glucose test kit (ASAN PHARMACEUTICAL, Seoul, Korea) and spectrophotometer (VersaMax microplate reader) And blood glucose was calculated.

The intraperitoneal glucose tolerance test (IPGTT) and the oral glucose tolerance test (OGTT) were performed at 4 weeks. The HFD group (11 weeks old) supplemented with normal diet (ND), HFD group, and DSW (1,000 and 2,000) were allowed to eat diets freely but restricted diets. Nepodin was orally administered at a rate of 2 mg / kg body weight and 10 mg / kg body weight at a time after 15 hours of fasting. Carboxymethyl cellulose alone (0.5 mL / 100 g body weight) was orally administered to m / m of the normal group and db / db of the diabetic group. Three hours later, blood was drawn from the tail vein of all rats and immediately all rats received an intraperitoneal glucose injection (0.2 g / mmL / 100 g body weight). Blood samples were taken consecutively at time intervals (0, 30, 60, 90 and 120 minutes) to measure blood glucose levels according to the method.

As a result, hyperglycemia and hyperglycemia were induced in experimental rats when compared with the group in which ND was fed. In the case of ingestion of minced water with a deep sea water mineral extract (hardness 500, 1,000 and 2,000), the increase in blood glucose was suppressed in a hardness-dependent manner (Fig. 1A) and the oral glucose tolerance test (OGTT ) And the abdominal glucose challenge (IPGTT) showed regression of glucose tolerance exacerbated by HFD in a dose-dependent manner (FIG. 1B). All figures in Figure 1 are expressed as mean + -standard deviation (n = 8 per group). * P <0.05, ** P <0.01: significant difference vs. HFD group.

Therefore, it has been confirmed that the deep sea water of the present invention has an excellent effect on inhibition of hyperglycemia and recovery of glucose tolerance.

Experimental Example  2: invention Deep sea water  Effect on Gene Expression Related to Glycogen Synthesis

In order to investigate the mechanism of hyperglycemia inhibition and glucose-lowering recovery effect of the deep sea water according to the present invention, in a liver which is one of important tissues for maintaining glucose homeostasis in the body, glycogenogenesis, glycogenesis, glycogen Gene expression related to glycogenolysis and glucose oxidation was investigated. RNA was isolated from liver and adipose tissue using Trizol (Invitrogen) and cDNA was synthesized using PrimeScript ™ 1st Strand cDNA Synthesis Kit (Takara). Real-time PCR was performed in the ABI Prism 7300 Sequence Detection System (Applied Biosystems) three times using FastStart SYBR Green Master mix (Roche). Expression of target genes was calculated proportionally using the delta cycle threshold method for the expression of endogenous standard gene actin. The primer sequences used are shown in [Table 2].

As a result, in the HFD group, liver glycogen phosphorylase (LGP) related to glucose 6-phosphatase (G6Pase), glycogen synthase (GS) related to glycogen synthesis (FIG. 2B) and glycogenolysis ), Glucokinase (GK) and citrate synthase (CS) related to glucose oxidation (Fig. 2C).

However, all of the genes except glycogen synthase (GS) were decreased in the HFD group ingested together with DSW of the present invention.

Therefore, it was confirmed that the deep sea water of the present invention can maintain blood glucose level of the general formula by inhibiting the increased glucose uptake, glycogenolysis, and glucose oxidative gene expression by the high fat diet method.

Experimental Example  3: invention Deep sea water  3 T3 - L1  Adipocyte To absorption of sugar  Effect

In order to examine the efficacy of the deep sea water of the present invention for glucose absorption, glucose uptake was examined using 3T3-L1 adipocytes. The glucose uptake by mature 3T3-L1 adipocytes was measured using 2-NDBG. Mature 3T3-L1 adipocytes were differentiated from 96 well black culture plates (Greiner Bio-One, CELL STAR 96W Palte, NC, USA) and washed with PBS. Cells were cultured in serum-free DMEM for 1 hour and then cultured in DMEM containing 10 μM of rosiglucarzon and 100 nM of insulin in the presence and absence of balanced DSW and 20 μM of 2-NDBG. After incubation for 1 hour, the cells were washed with PBS and incubated with lysis buffer (0.1 M potassium phoshate, 1% Triton X-100, pH 10) for 10 minutes with agitation. Dimethyl sulfoxide (DMSO) was added while stirring for another 10 minutes. Fluorescence signals were measured at a 466 nm excitation wavelength and a 540 nm emission wavelength using a microplate reader (FLUOstar OPTIMA, BMG LABTECH, Germany).

After the cells were dissolved, glucose uptake was measured using a fluorescence intensity meter, and all values were expressed as mean ± standard deviation. * P <0.05, ** P <0.01: significant difference vs. Untreated group (CON)

 As a result of comparing the glucose uptake of the deep sea water treated groups according to the present invention (by hardness) and the glucose uptake enhancing group with respect to the untreated group (CON), the glucose uptake in the deep sea water treated group was increased in the degree of hardness. As can be seen in FIG. 3, the effects of glucose absorption by rosiglitazone and insulin, which are already known as glucose uptake enhancers of adipocytes, were observed at a level similar to that of glucose up to 1,500.

Therefore, the present invention suggests that deep sea water increases the glucose uptake efficiency in adipose tissue, thereby suppressing hyperglycemia by the high fat diet.

Experimental Example  4: invention Deep sea water AMPK  Phosphorylation and To absorption of sugar  Effect

To investigate which signaling substance is involved in glucose uptake by the deep sea mineral extract of the present invention, 3T3-L1 adipose precursor cells were differentiated into adipocytes for 8 days in a medium containing DSW according to hardness, SDS-PAGE and western blotting analyzes were performed using anti-phospho-AMPK, anti-phospho-ACC1 and anti-phospho-IRS-1AMPK antibodies as cell lysates.

Cells were washed with ice-cold PBS, scraped with ice and then dissolved in RIPA buffer. Total cellular proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membranes were blocked with 5% skimmed milk for 1 hour and incubated overnight at 4 ° C with anti-phosphoAmpK, anti-phospho IRS-1, anti-GLUT4 and anti-beta actin antibody (diluted 1: 3,000). After washing with TBS containing 0.1% Tween-20, the membrane was incubated with an oxidoreductase conjugated secondary antibody (1: 3,000) at room temperature for 1 hour. Antibodies bound to the nitrocellulose membrane were detected by ECL and radiography.

As a result of the experiment, it was confirmed that the activity of AMPK and ACC1, which is a substrate of the AMPK, is increased by deep seawater depending on the hardness (FIG. 4A). In addition, the deep sea water of the present invention increased IRS-1 activity of the insulin signal transduction system, which is one of the major glucose uptake signal transduction systems, and expression of GLUT4 directly responsible for glucose uptake in a hardness-dependent manner (FIG.

Therefore, it is suggested that the deep sea water of the present invention can increase the glucose uptake by activating the glucose uptake related signal transduction system.

Statistical analysis

All experimental results were compared by one-way analysis of variance (ANOVA) using the SPSS (ver.11.0) program and the experimental data were expressed as mean ± SD. Group ANOVA was considered significantly different at P <0.05 as determined by the description of the least significant difference protected when showing overall significant treatment outcomes at P <0.05.

Industrial availability

The present invention is an extremely useful invention in the health functional food industry and the biomedical industry because it has an excellent effect of providing deep sea water mineral extract having an effect of restricting blood sugar increase and restoring glucose tolerance.

Claims (5)

A pharmaceutical composition for prevention and treatment of hyperglycemia and obesity type 2 diabetes mellitus comprising a deep sea water mineral extract having a weight ratio of magnesium to calcium of 3: 1 and a hardness of 1,000 to 2,000 ppm as an active ingredient. A composition for the prevention and improvement of diabetes mellitus of hyperglycemia and obesity type 2, which comprises as an active ingredient a deep sea water mineral extract having a weight ratio of magnesium to calcium of 3: 1 and a hardness of 1,000 to 2,000 ppm. delete The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is any one selected from the group consisting of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral preparations, external preparations, suppositories and injections. A pharmaceutical composition for the prevention and treatment of obesity type 2 diabetes. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Characterized in that it further comprises a carrier selected from the group consisting of microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil and an excipient. A pharmaceutical composition for the prevention and treatment of type 2 diabetes.

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