KR20170043466A - Manufacturing method of anti-obesity solar salt and pharmaceutical composition comprising thereof - Google Patents

Abstract

The present invention relates to a functional anti-obesity composition containing bay salt as an active ingredient. Since the composition exhibits anti-obesity functions, it is possible to produce functional anti-obesity foods or pharmaceutical compositions for preventing and treating obesity containing the same as the active ingredient.

Description

[0001] The present invention relates to a method for producing an anti-obesity functional silver salt and a pharmaceutical composition for treating or preventing obesity containing the same,

The present invention relates to a process for producing a salt of sun salt having an anti-obesity effect and a pharmaceutical composition for treating or preventing obesity containing the same as an active ingredient.

Obesity is defined as 'a state in which body fat accumulates excessively above normal' in modern medicine, and over one billion people were diagnosed as obese. In particular, two-thirds of US adults are obese, and the majority of obese and overweight people in other developed countries are not obese compared to the developed countries in Korea. However, after increasing from 26.0% in 1998 to 31.3% in 2005, The obesity rate is 32%. Obesity is a major cause of global health problems that cause metabolic diseases such as hyperlipidemia and diabetes.

Obesity and its complications are controlled by lipid metabolism related proteins. Current treatments for treating obesity include drugs that act on the central nervous system, affect appetite, and drugs that act on the gastrointestinal tract to inhibit absorption. Drugs such as fenfluramine and dexfenfluramine which inhibit the serotonin (5HT) nervous system, drugs such as ephedrine and caffeine through the noradrenergic nervous system, and recently, drugs acting on the serotonin and noradrenergic nervous system And sibutramine, which acts to inhibit obesity. Other drugs that inhibit obesity by acting on the gastrointestinal tract include orlistat, which has recently been approved as a treatment for obesity by inhibiting lipase produced by the pancreas and reducing fat absorption. However, of the existing obesity treatments, fenfluramine has been recently banned due to side effects such as primary pulmonary hypertension or heart valve lesions. Sibutramine has already been withdrawn from the market due to side effects of increasing blood pressure. Side effects are known. In addition, other chemical synthetic drugs have problems such as decreased blood pressure and lactic acidosis, and thus they can not be used for patients suffering from heart failure or renal disease.

Leptin is a protein composed of 167 amino acids transcribed from the ob gene and is recognized as the first adipokine (Pelleymounter MA, Cullen MJ, Baker MB, Hecht R, Winters D, Boone T, Collins F. Effects of the obese gene product on Localization of the human OB gene (OBS) to chromosome 7q32 < RTI ID = 0.0 > (I) < / RTI > by fluorescence in situ hybridization Genomics 28: 603-604, 1995; Saladin R, De Vos P, Guerre-Millo M, Leturquee A, Girard J, Staels B, Auwerx J. Transient increase in obese gene expression after food intake or insulin administration. Nature 377: 527-529, 1995). Leptin is known to induce satiety in the hypothalamus of the hypothalamus on the brain-gut axis (Konturek PC, Brzozowski T, Burnat G, Kwiecien S, Pawlik T, Hahn EG, Konturek SJ. Role of brain-gut axis In addition, we found that the paclitaxel secretion and appetite control of the brain-gut axis in J, G, and J, Physiol Pharmacol 54: 293-317, 2003), genetically deficient adults with leptin have elevated appetite and over-induction of obesity, as well as hyperinsulinemia, insulin resistance, hyperlipidemia, immunodeficiency, and endocrine disorders There are reports (Farooqi IS, Matarese G, Lord GM, Keogh JM, Lawrence E, Agwu C, Sanna V, Jebb SA, Perna F, Fontana S, Lechler RI, DePaoli AM, O'Rahilly S. Beneficial effects of leptin on obesity, T cell hyporesponsiveness, and neuroendocrine / metabolic dysfunction of human congen ital leptin deficiency. J Clin Invest 110: 1093-1103, 2002; Montague CT, Farooqi IS, Whitehead JP, Soos MA, Rau H, Wareham NJ, Sewter CP, Digby JE, Mohammed SN, Hurst JA, Cheetham CH, , Barnett AH, Prins JB, O'Rahilly S. Congenital leptin deficiency is associated with severe early-onset obesity in humans. Nature 387: 903-908,1997). It is also reported that leptin receptor mRNA and protein expression increases on atherosclerotic plaque by feeding high fat diet, and luminal stenosis is detected when leptin is injected into ob / ob mouse (Schafer K, Halle M, Goeschen C , Dellas C, Pynn M, Loskutoff DJ, and Konstantinides S. Leptin promoting vascular remodeling and neointimal growth in mice. Arterioscler Thromb Vasc Biol 24: 112-117, 2004) Jones JH, Johnson O, Hallmans G, Asplund K, Olsson T. Leptin is associated with increased risk of myocardial infarction. J Intern Med 246: 409-418, 1999; Reilly MP, Iqbal N, Schutta M, Wolfe ML, Scally M, Localio AR, Rader DJ, Kimmel SE.Plasma leptin levels are associated with coronary atherosclerosis in type 2 diabetes.J Clin Endocrinol Metab 89: 3872-3878, 2004).

Salt accounts for about 0.9% of the blood of human body, and it is a component of various body fluids such as various digestive juices and lymphatic fluid, and is the most important substance in controlling osmotic pressure. In addition, it is a biomodulatory substance that is involved in physiological functions such as muscle contraction and acid-base equilibrium. It is an essential substance for human life (Bae D. H. Hazardous contaminants in commercial salts, Safe Food, 4, 14-24, 2009). Salty salt, salt, and sea water are the raw materials used to consume salt, which is an essential substance, and seawater is the largest salt reservoir on earth, and its amount is large, which is easily obtained. The edible salt is divided into the salt of the sun and the salt of the salt, and the salt of the salt is obtained by the stepwise concentration of seawater by introducing seawater into the reservoir by using nature such as solar heat and wind (Hwang SH A study on the heavy metal contents of common Salt refinement is divided into mechanically produced mechanically salted salted salted roasted salt and bamboo salted salted salted salted salted salted salted salted salted salted salted salted salted carrots Ha JO et al., Comparison of mineral contents and various structures of various salts, Korean J. Food Sci., Nutr., 27, 413-418, 1998). Silver salts are more abundant in inorganic salts such as NaCl, K, Mg, Ca and S than in tablets, and Gerald salt in France is famous for high-quality silver salt. The salt prepared by dissolving the raw material salt in purified water, seawater or sea water concentrate, filtration, precipitation, recrystallization, dehydration, and salting is referred to as refined salt, and the raw salt is converted into salt, Salt, refined salt and other salt are processed salt with 50% or more of food or food additives (KFDA, Food code. Korean Food & Drug Administration, Seoul, Korea, P19-23, 2011).

Prior art for the anti-obesity composition includes hydroxamic acid derivatives showing anti-obesity effects and anti-obesity or body fat reducing agents containing MeJA-treated buckwheat sprouts as disclosed in Japanese Patent Application No. 10-0845511 The composition is disclosed in Korean Patent No. 10-1282708. An anti-obesity composition containing an acacia bark extract is also disclosed in Korean Patent No. 10-1369060. However, none of the above documents teach or disclose pharmaceutical compositions for treating or preventing obesity that include sun-salt and an effective ingredient thereof, which have an anti-obesity effect.

Accordingly, it is an object of the present invention to provide a method for producing a salt of sun salt having an anti-obesity effect.

It is still another object of the present invention to provide a pharmaceutical composition for treating or preventing obesity, which comprises an effective ingredient of sun salt having an anti-obesity effect.

The object of the present invention is to provide a method for the treatment of seawater, comprising: (a) introducing seawater having a salinity of 1 to 2 degrees into a reservoir; (B) a step of transferring the seawater stored in the reservoir to the first evaporation paper (incompetent) and evaporating the same for one week to obtain an egg shell having a salinity of 6 to 8 °; (C) transferring the wastewater obtained in the step (b) to a second evaporation paper (nest) and evaporating the wastewater for one week to obtain a wastewater having a salinity of 14 to 18 °; (D) storing the wastewater obtained in the step (c) in a warehouse for 25 days to obtain a brine having a salinity of 23 to 25 °; (E) storing salt water obtained in step (d) in a crystal paper to saturate the salt crystals at a salt concentration of 27 °; (F) a step of supplying salt crystals by supplying a function of salinity of 22 to 23 ° to the first crystallized paper in the step (e), and further feeding (pouring) a function of salinity of 25 ° to the determined paper for 4 days; The salt obtained in the steps (e) and (f) is stored in a salty storage and about 80% of the water is removed to obtain a salt. The solar salt obtained in the above step is used as a test material in C57BL / 6 mouse After the induction of obesity using a model, obesity was induced for 8 weeks by dividing into chow diet, high fat dietary group (HFD), refined salt combination diet group, and sun salt combination dietary group and then replaced with high fat diets containing salt The effects of dietary supplementation on body weight gain, dietary efficiency, liver weight change, testosterone weight change, triglyceride content change, total cholesterol content change, leptin content change, insulin resistance evaluation, liver histopathology effect, adipogenic / lipolytic factor, and inflammatory factor in the rat brain.

The present invention has an excellent effect of providing a suntan salt having an anti-obesity effect and a pharmaceutical composition for treating or preventing obesity containing the same as an active ingredient.

1 is a flow chart showing the sequence of manufacturing process of the sun salt.
Fig. 2 (a) is a photograph showing a reservoir for storing seawater having a salinity of 1 to 2 degrees.
Fig. 2 (b) is a photograph showing the gates and waterways of the reservoir.
Fig. 3 is a photograph showing an environmentally friendly first evaporation paper (hard-edged;
4 is a photograph showing a second evaporation paper (slowness: 5 to 7 stages).
Fig. 5 is a photograph showing a roof (functional warehouse) covered with a non-occlusive material with an eco-friendly material.
Fig. 6 is a photograph showing a determination paper (1 to 4), which is a space in which a floor is covered with a cover plate and is in a container.
FIG. 7 is a photograph showing a sleeping passage, which is a passage through which the remaining jug (31 ~ 32 °) left after the flushing is mixed with the flushing water.
FIG. 8 is a photograph showing a structure in which the function transfer of the hand is rapidly performed using an underwater electric motor pump and a hose. FIG.
FIG. 9 (a) is a photographic view outside the warehouse storing the salt salt (20% moisture saturation degree).
FIG. 9 (b) is a photograph of the inside of a salt warehouse dried to allow high quality (15% moisture, milk light) salt to be aged by dripping the salt.
FIG. 10 is a graph showing weight gain of mice in the formulation of the sun salt formulation. FIG.
FIG. 11 is a graph showing the dietary efficiency of the mouse with the salt formulation.
FIG. 12 is a graph showing changes in liver weight of mice in the formulation of the sun-salt.
13 is a graph showing the change in the weight of testis fat of mice in the formulation of the sun-salt.
FIG. 14 is a graph showing the change in the triglyceride content of mice of the present invention and the other formulation of the sun salt formulation.
15 is a graph showing changes in total cholesterol content of mice in the formulation of the sun-salt.
16 is a graph showing the change in leptin content of mice in the formulation of the sun-salt.
FIG. 17 is a graph showing the insulin resistance of mice in the formulation of the sun salt formulation.
18 is a graph showing the hepatic histopathology effect of mice in the formulation of the sun-dried salt.
FIG. 19 is a graph showing the adipogenic pathology effect of mice in the formulation of the sun salt formulation. FIG.
FIG. 20 is a photograph showing the change in the expression level of adipogenic / lipolytic factor in the liver tissue of mice in the formulation of the sun salt formulation.
FIG. 21 is a photograph showing the change in expression level of inflammatory factor in the liver tissue of mice in the formulation of the sun salt formulation. FIG.

According to the present invention, the most preferred process for producing a salt of the sun salt comprises the steps of (a) introducing seawater having a salinity of 1-2 ° into a reservoir; (B) a step of transferring the seawater stored in the reservoir to the first evaporation paper (incompetent) and evaporating the same for one week to obtain an egg shell having a salinity of 6 to 8 °; (C) transferring the wastewater obtained in the step (b) to a second evaporation paper (nest) and evaporating the wastewater for one week to obtain a wastewater having a salinity of 14 to 18 °; (D) storing the wastewater obtained in the step (c) in a warehouse for 25 days to obtain a brine having a salinity of 23 to 25 °; (E) storing salt water obtained in step (d) in a crystal paper to saturate the salt crystals at a salt concentration of 27 °; (F) a step of supplying salt crystals by supplying a function of salinity of 22 to 23 ° to the first crystallized paper in the step (e), and further feeding (pouring) a function of salinity of 25 ° to the determined paper for 4 days; The salt obtained in the steps (e) and (f) is stored in a salt warehouse and about 80% of the wastewater is removed to obtain a salt having a Na / K ratio of 18 to 35.

In the present invention, CHOW is a general dietary group, HFD is a high-fat dietary group, PS is a combination dietary group containing tablets, SS-Y is a combination dietary group containing dietary supplements, SS- SS-S1 is a combination formula containing salty salt, SS-S2 is a combination formula containing coarse salt before Shin-il salt, SS-G is a combination formula containing gerendotopan salt, SS Is a group of Buan gomgok tidal formula.

Hereinafter, the present invention will be described in more detail with reference to embodiments and drawings. However, the following examples are intended to illustrate the invention and are not intended to limit the scope of the invention.

< Example  1> Method for manufacturing solar-salt

The method of manufacturing the salt of the present invention was carried out in Jeungdo-myeon, Sinan-gun, Chonnam, and Dochon-myeon, Chunnam-gun, Jeonnam, and the concrete example was completed for 214 days from April 1, 2015 to October 30, Was performed using the salt of the salt produced in the torsion as the disclosure material.

The schematic process of the method of manufacturing the present invention and the method of producing the salt is shown in Fig. First, in order to introduce seawater (salinity 1 ~ 2 °) into a tidal reservoir at high tide, the water intake (Figure 2b) is opened and seawater is introduced into the reservoir (Figure 2a).

The seawater stored in the reservoir (FIG. 2 a) is sent to the first evaporation paper (FIG. 3 a) of 8th to 11th steps of 8 steps, also called inconvenient, The water is evaporated, and the brine is supplied with salinity of 3 ~ 8 ° according to the sunlight. The saline solution is sent to the second evaporation paper (Figs. 4a to 4b) of 7th to 5th steps, which is also referred to as "Zelt" At a salt content of 14 to 18 DEG, preferably at least 15 DEG C., is produced.

In the reservoir, the seawater (Boomer 1 ~ 2) of the first evaporation paper is settled in the middle of the 4th stage. It is considered that the present invention increases the content of minerals such as K and Ca (SS-T, SS-S1 salt). The depth of the eleventh stage of the first evaporation paper is 10 cm, and the width is narrowed down from the 10th stage and the depth of water is decreased by about 1 cm. If the color of the salt is milky and the size of the salt crystals is large, the winter cover is effective (SS-S2 salt). The second evaporator consists of approximately three stages in five to seven stages. In this case, particularly insoluble components are precipitated.

At this time, a function warehouse (hereinafter also referred to as an underground salt storehouse) of the second evaporation sheet is installed in approximately 5 to 6 places to store the salt water by salinity (FIGS. 5A to 5B). The function warehouse is constructed and installed adjacent to the determination site (Figs. 6 (a) to 6 (b)) in which the number and size of the warehouse are determined according to the size of the torsion springs. The salinity of the brine is kept at 23 ~ 25 °. In the second evaporation paper (Figs. 6a to 6b), the water depth is adjusted to adjust the saturation function to be supplied with the crystallization paper at the shortest (called a rice nucelle).

That is to say, (4) is a function storage space (upper part in FIG. 7 (a)) in which a high concentration function is stored. The roof of the building is made of a non-greasy material and eco-friendly materials are used in consideration of food hygiene. 6a to 6b), a salt flower having a salinity of 27 degrees in a saturated state is formed after a certain period of time, and the salt flowers are gathered together to form small grains And the salt crystals are formed with the cube, and it is possible to accumulate at about 4 ~ 6 pm. Generally, the salt produced on the day is weak and easily crumbled, and the daily production amount is usually about 1,500 kg (1.5 ton) per information in the 5th row and 6th row of the final determination paper (Fig. 7a to Fig. 7b).

If salt of solid crystal is to be collected, salt crystals are formed when a salt flower is formed and settled by supplying a function of salinity of 22 ~ 23 ° through the water pipe (3) of (4) When the other 25 ° function is added to the decision paper and the addition is continued for 2 ~ 3 days, the strength of the salt crystals is increased. However, the yield of production is reduced. In order to obtain salt crystals with enhanced strength in this way, it is possible to harvest about 1,125 kg (1.125 ton) per information in the four days after collection.

On the other hand, a function water pipe 5 and a valve 6 are installed for efficiently spreading the water of the water hose 4 all the air (Figs. 8A to 8B). In this way, salt is produced in the final determination ground, and the bottom 7 of the determination grounds 7a to 7b as a collection space has the form of a pottery plate, a top plate, a long plate, and a tile plate.

In general, the mixture is stored at the interval of 30 ~ 32 ° in the remaining 30 ~ 32 ° intervals, and stored 14 ~ 17 ° in the ground. After that, it is cleaned in the morning or the next morning and the water is put into the decision paper again to wait for the salt crystals. If the saturated function 25 ° is sufficient, the water is removed through the column 8, and if it is not sufficient, the function of 14-17 ° at the bottom of the evaporation paper is mixed with 23 ~ 25 ° so that the impurities are precipitated by flowing into the crystal blank.

The thickness of the crystals (7a ~ 7b) is preferably 1.5 ~ 2cm in summer and 1 ~ 1.5cm in spring and fall in the case of bottom plate, and it is supplemented by 26 ~ ° so that the salt crystals are precipitated. According to the present invention, about 90% of salt is precipitated up to 30 ° bore and the Mg content is rapidly increased from 31 ° bore to deteriorate the salt quality.

The salt thus produced is removed from the salt water (MgCl ₂ ) and transferred to the salt warehouse 9a so that water is dripped and aged. The moisture content of the salt collected in the decision site is more than 20%. And the moisture content is about 15% when the aging period is over three months. In addition, the magnesium chloride is eluted, the bitter taste disappears, and it is matured with soft, light milk high quality salt. The bottom of the salt warehouse 9a is constructed of wood and is designed to be installed over the entire surface of the warehouse floor and to be discharged to the bottom floor through the central water channel 9. [ Finally, the salinity of the aged salt is greatly reduced to 86 ~ 89% from 73 ~ 85% of the salinity of the sand.

The weight gain of mice raised with the salt (SS-T) prepared in the Taepyeong tidal field and the salt (SS-S1 and SS-S2) prepared with Shinil tidal salt according to the present invention was shown in FIG. 10 Appeared to be effective in suppressing obesity.

< Example  2> The present invention relates to a method for producing a compounded diet containing sun salt

A salt sample such as the salt of the present invention prepared according to Example 1 was mixed with a high-fat diet (D12451; 45% calories from fat) to prepare a test material. The composition of the test sample is shown in Table 1 below. HFD + salt was mixed with different kinds of salt to make salt mixture.

The mixing ratio of the sun-salt mixed feed according to the present invention Ingredient
(g / 1000 g diet) Groups Normal HFD HFD + Salt Casein 200 245 244 L-Cystine 3 4 4 Corn Starch 397.5 85 85 Maltodextrin 132 115 114 Sucrose 100 200 199 Lard - 195 194 Soybean oil 70 30 30 Cellulose 50 58 58 Mineral mix
(AIN-93GMX) 35 43 43 Vitamin mix
(AIN-93VX) 10 19 19 Calcium phosphate, Dibasic - 3 3 Choline Bitartrate 2 3 3 Red Food color - 0.1 - Salt samples ⁽¹⁾ - - 4.7 Total calorie (cal / g) 3800 4600 4578

< Experimental Example  1> Analysis of mineral content of sun salt

The inorganic composition of the sodium salt samples used in this experiment was analyzed by ICP-OES method. As a result, the mineral content of domestic sun - salt was found to be superior to that of Gurand salt as well as refined salt. In particular, the SS-T of the present invention has a high content of potassium and calcium, and the SS-S1 of the present invention has the lowest Na / K ratio (Table 2).

Mineral content of sun salt content (%) PS SS-T SS-Y SS-S1 SS-S2 SS-G Na 38.9031 30.3086 57.9796 34.6106 30.6790 37.8776 Mg 0.0199 1.3215 1.5737 1.2569 0.5910 0.2271 K 1.0117 1.6213 1.9847 1.9427 0.8795 0.3041 Ca 0.0232 0.2388 0.0674 0.1060 0.0726 0.1567 S 0.0101 1.0313 1.0091 0.7304 0.3370 0.2533 Zn 0.000 0.000 0.000 0.000 0.000 0.000 Pb 0.000 0.000 0.000 0.000 0.000 0.000 CD 0.000 0.000 0.000 0.000 0.000 0.000 As 0.000 0.000 0.000 0.000 0.000 0.000 Na / K 38.45436 18.69378 29.21391 17.81594 34.88284 124.5699

^{[Note] PS: refined salt, SS-Y: Amomum villosum, SS-T: Pacific salt, SS- S1: Shinil torsion going salt, SS- S2: Shinil salt coarse salt, SS-G: gerang} de tryptophan salt

< Experimental Example  2> According to the present invention, Anti-obesity  Verification of effect

80 dogs of C57BL / 6 mice were divided into two groups: chow diet, high fat diets (HFD), refined salt combination diets, and sun salt combination diets according to the present invention. After one week of refinement, all experimental groups except the normal diet group were supplemented with 40% fat high fat diet (D12450) for 8 weeks and then supplemented with high fat diet containing salt for an additional 8 weeks . After the administration, autopsy was carried out without fasting, and blood sampling and organ harvesting were carried out at autopsy (Proc Natl Acad Sci USA, 95, P5987-5992, 1998).

Blood was drawn from the abdominal vein. The blood was immediately stored at 4 ° C and centrifuged at 13,000 rpm at 4 ° C to separate the serum. The collected serum was used to measure the content of TG, total cholesterol and HDL cholesterol, and was also used for cytokine analysis by ELISA and the like.

Organ harvesting was done by weighing testis fat and liver and fast freezing with liquid nitrogen.

(1) Weight gain

As shown in FIG. 1, the body weight of the present invention SS-T and SS-S1 were 21.6 and 21.3 grams, respectively, which was significantly lower than that of HFD (26.5 grams) and SS-G (25.1 grams) appear.

(2) Dietary efficiency

As shown in FIG. 2, the dietary efficiency of SS-S1 and SS-T according to the present invention was significantly lower than that of SS-G in the HFD group.

(3)

As shown in FIG. 3, the weight of liver was significantly increased in the experimental group compared to the control (normal diet group), and the weight of SS-S1 and SS-S2 among the salt formulations of the saline group was 2.13 and 2.14 grams And the lowest among the invented astringent group.

(4) Symptomatic  Weight change

As shown in FIG. 4, the weight of test fat in the HFD group was significantly increased in the experimental group as compared to that in the control group (normal diet group), and that of the SS-S1 and SS-S2 The weight of testis fat was 1.53 and 1.55 grams, respectively, indicating a significant decrease compared to the HFD group.

(5) Change in triglyceride content

The triglyceride content of the serum phase was measured using a cleansing reagent (Cleantech TG-S) from Asan Pharmaceutical Co., and 20 μl of serum, standard solution or distilled water was added per 3 ml of the prepared enzyme solution, and the mixture was incubated at 37 ° C. for 10 minutes. The absorbance is measured at 550 nm within 60 minutes. A linear calibration curve is constructed based on the absorbance at 0 mg / dl and 300 mg / dl, and the absorbance of the reagent reacted with the serum is converted and converted.

As shown in FIG. 5, the triglyceride content of the serum was significantly higher in the whole experimental group than in the HFD group. However, the neutral fat content of the salted salted salted and salted Salted salted salted salted salted salted salted salted salted salted salted salted salted salted salted salted salted salted salted salted salted salted salted salted salted salted salted salted salmon.

(6) Changes in total cholesterol content

The total cholesterol content of the serum phase was measured by using T-CHO for the determination of triglyceride in Asan Pharmaceutical Co., and 20 μl of serum, standard solution or distilled water was added per 3 ml of the prepared enzyme solution, and the mixture was incubated at 37 ° C for 5 minutes. After that, the absorbance is measured at 500 nm within 60 minutes. A linear calibration curve is constructed based on the absorbance at 0 mg / dl and 300 mg / dl, and the absorbance of the reagent reacted with the serum is converted and converted.

As shown in FIG. 6, the total cholesterol content was 163.39 mg / dl in the SS-T, which was the lowest in the group treated with the saline, and 165.09 mg / dl in the SS-S1 according to the present invention. On the other hand, SS-G showed 205.35 mg / dl, which is significantly higher than HFD.

(7) Leptin  Content change

Serum Leptin concentrations were determined using a Quantikine ELISA kit from R & D Systems (Minneapolis, MN, USA). 50 μl of assay diluent was added to the wells to which the primary antibody had been pre-transfected. Then, pre-prepared standard calibrator-specific leptin solutions (2000, 1000, 500, 250, 125, 62.5, 0 pg / Of serum are added to each well and stirred at room temperature for 2 hours. After washing 4 times with wash buffer, 100 μl of leptin detection antibody solution is added and stirred at room temperature for 2 hours. After washing 4 times with wash buffer, 100 μl of substrate solution is added and stirred for 30 minutes in the dark. After completion of the reaction, 100 μl of stop solution is added and the absorbance is measured at 450 nm within 30 minutes. Prepare a calibration curve using the absorbance of the portion containing the calibration curve solution, and then convert the absorbance of the reagent reacted with the serum.

As shown in FIG. 7, Leptin showed lower levels of leptin than the HFD group. Especially, 3.1ng / ml for SS-T and 3.31ng / ml for SS-S1 were found to be significantly lower in the whole group.

(8) Evaluation of insulin resistance

Serum insulin concentrations are calibrated using a special kit from Millipore (St. Charles, Missouri, USA). The wells are washed three times with 300 μl wash buffer, and then 10 μl of assay buffer (20 μl in blank wells and 10 μl in remaining wells) is placed. Then, 10 μl of the serum of each test group was added to each well of the standard insulin solution (0.2, 0.5, 1, 2, 5 ng / ml) prepared beforehand and 80 μl of detection antibody was added. . After washing with wash buffer 3 times, 100 μl of enzyme solution is added and stirred at room temperature for 30 minutes. After washing with wash buffer 6 times, 100 μl of substrate solution is added and stirring is carried out for 5-20 minutes at room temperature. If the color tone of the calibration curve shows several stages of blue color, 100 μl of stop solution is added and absorbance is measured at 450 nm under 5 minutes . Prepare a calibration curve using the absorbance of the portion containing the calibration curve solution, and then convert the absorbance of the reagent reacted with the serum.

The concentration of glucose in the blood was measured by the reagent for measuring glucose in Asan (AM201K). 3 ml of enzyme reagent was added to 20 μl of serum, distilled water and standard solution, and the mixture was incubated at 37 ° C. for 5 minutes. Then, absorbance was measured at 500 nm within 30 minutes. Construct a linear calibration curve based on the absorbance of the distilled water group and the standard solution group, and calculate the absorbance of the reagent reacted with the serum.

As shown in FIG. 8, the HOMA-IR level of the HFD-treated group was increased to 2.85 as compared with the control (1.15), whereas the HOMA-IR level of the treated group was lower than that of the control group (1.15). Among them, SS-S1-treated group had 2.16, which was the lowest among the other treatments.

(9) Liver tissue  Pathological effect

Fragments of mouse liver were fractionated and H & E staining was performed, and lipids in the tissues were observed with an electron microscope.

As a result, as shown in FIG. 9, compared with the liver tissues of the Chow diet group, the fat tissues of the HFD group were relatively larger than those of the Chow diet group, and the fat tissues of the group of the refinement formula were slightly smaller than those of the Chow diet group. On the other hand, the scale of SS-T and SS-S1 showed the least amount of fatty waters in comparison with those of other saline-treated groups.

(10) adipose tissue ( Essential oil ) Pathological effect

The extracted adipose tissue was fragmented and H & E stained, and the tissue was observed with an electron microscope.

As a result, as shown in FIG. 10, the fatty tissue of the HFD group was relatively larger than that of the fat tissue of the Chow diet group, and the fat area of the group of the purified formula was slightly smaller than that of the fat tissue of the Chow diet group. On the other hand, in the case of the salt-in-salt formula group, the scale of the fat ward in the SS-S1 was shown to be nearly as high as the chow diet. In addition, macrophage infiltration was observed in the adipose tissue during induction of obesity, and the PS level was higher than that of the HFD group. In the present study, macrophage infiltration in the SS-T and SS- As shown in Fig.

11, the expression level change in the adipogenic / lipolytic factor on liver tissue according to the present invention administered solar salt

Obesity related factors were observed by PCR in liver tissue. The liver tissues were immersed in TriZol buffer (Invitrogen, Carlsbad, Calif.), And homogenized with a homogenizer. The first strand cDNA was synthesized using Superscript II reverse transcriptase (Invitrogen, Carlsbad, Calif.) And applied to RT-PCR. Amplification was performed by repeating 40 cycles of incubation at 94 ° C for 1 min, annealing at 54 ° C for 1 min, extension at 72 ° C for 30 sec, and termination by incubation at 72 ° C for 7 min . The amplified product was electrophoresed on a 1.0% agarose gel containing ethidium bromide (Et-Br) and the product was quantitated under UV light.

As a result, as shown in FIG. 11, the expression of PPAR-γ, a factor associated with adipogenesis, was strongly inhibited in SS-S1 of the present invention. FAS, which is a factor directly involved in lipogenesis, Expression was suppressed. On the other hand, CPT-1, a factor related to β-oxidation, was found to be exaggerated in SS-S2 in the saline-treated group.

(12) The expression level of inflammatory factor in liver tissue by administration of the sun salt of the present invention

Inflammatory response factors were observed by PCR in liver tissue. Liver tissues were immersed in a TriZol buffer (Invitrogen, Carlsbad, Calif.) As described in (11) of the present invention <Experimental Example 2>, followed by pulverization with a homogenizer. The first strand cDNA was synthesized using Superscript II reverse transcriptase (Invitrogen, Carlsbad, Calif.) And applied to RT-PCR. Amplification was performed by repeating 40 cycles of incubation at 94 ° C for 1 min, annealing at 54 ° C for 1 min, extension at 72 ° C for 30 sec, and termination by incubation at 72 ° C for 7 min . The amplified product was electrophoresed on a 1.0% agarose gel containing ethidium bromide (Et-Br) and the product was quantitated under UV light.

As a result, as shown in FIG. 12, the expression level of SS-S1 was the lowest in TNF-a directly involved in the inflammation of the tissues in the group treated with sodium chloride of the present invention. In addition, MCP-1, which is involved in the aggregation of macrophages and induces an inflammatory response, was also inhibited in SS-S1.

In conclusion, SS-S1 of the present invention has a mechanism of inhibiting lipid synthesis while enhancing β-oxidation by acting directly on adipocytes. This resulted in inhibition of weight gain in mice, improvement of lipid metabolism, and inhibition of obesity.

As described above, the present invention has an excellent effect of providing an anti-obesity composition containing sun-salt, so that it is an extremely useful invention in the diet functional food industry and the pharmaceutical industry for the prevention and treatment of obesity by providing an anti- .

Claims (4)

(A) introducing seawater having a salinity of 1 to 2 占 into the reservoir; (B) a step of transferring the seawater stored in the reservoir to the first evaporation paper (incompetent) and evaporating the same for one week to obtain an egg shell having a salinity of 6 to 8 °; (C) transferring the wastewater obtained in the step (b) to a second evaporation paper (nest) and evaporating the wastewater for one week to obtain a wastewater having a salinity of 14 to 18 °; (D) storing the wastewater obtained in the step (c) in a warehouse for 25 days to obtain a brine having a salinity of 23 to 25 °; (E) storing salt water obtained in step (d) in a crystal paper to saturate the salt crystals at a salt concentration of 27 °; (F) a step of supplying salt crystals by supplying a function of salinity of 22 to 23 ° to the first crystallized paper in the step (e), and further feeding (pouring) a function of salinity of 25 ° to the determined paper for 4 days; Wherein the salt obtained in the steps (e) and (f) is stored in a salt warehouse, and about 80% of the wastewater is removed. Wherein the Na / K ratio is in the range of 18-35. 8. An anti-obesity functional food composition comprising the sun-salt of claim 2 as an active ingredient. A pharmaceutical composition for the prevention and treatment of obesity comprising the sun-salt of claim 2 as an active ingredient.

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