KR101095035B1 - Composition for the prevention and treatment of obesity, hyperlipidemia and fatty liver comprising Zingerber officinale, Taraxacum platycarpum and Scutellaria baicalensis - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention and treatment of obesity, hyperlipidemia and fatty liver, including pogongyoung, ginger and gold in an optimal ratio, and to a dietary supplement composition for the prevention and improvement of obesity, hyperlipidemia and fatty liver, The mixture of ginger and gold has excellent inhibitory activity of lipolytic enzymes, effectively inhibits weight gain due to high fat diet, significantly inhibits the size of fat cells, and can be useful for treating obesity, and reduces blood lipids. It is also effective in improving and treating hyperlipidemia due to obesity, and reducing total cholesterol, effectively improving serum lipid level, reducing liver tissue lipid level, and inhibiting the deposition of fat globules in liver tissue, which can contribute to fatty liver improvement. It has an effect.

Description

Composition for the prevention and treatment of obesity, hyperlipidemia and fatty liver comprising Zingerber officinale, Taraxacum platycarpum and Scutellaria baicalensis}

The present invention relates to a composition for preventing and treating obesity, hyperlipidemia and fatty liver, including pogongyoung, ginger, and gold, and more specifically, an agent for preventing and treating obesity, hyperlipidemia and fatty liver, including pogongyoung, ginger, and gold in an optimal ratio. The present invention relates to a pharmaceutical composition and a dietary supplement for preventing and improving obesity, hyperlipidemia and fatty liver.

Recently, with the development of the industry and the improvement of income level and the westernization of lifestyles such as eating habits and eating habits, chronic diseases and adult patients are rapidly increasing, and one of the foundations is known as obesity. The scope of obesity is not limited only to adults, but recently, the problem has been added to childhood obesity and adolescent obesity, and the severity is increasing. The Korean health association has already defined obesity as a chronic disease. I am interested in

Obesity refers to a condition in which abundant adipose tissue is accumulated in the body. The most common cause of obesity is weight gain or body fat accumulation due to ingestion of high energy or high fat foods and lack of exercise. It is also reported to be caused by systemic abnormalities, drugs, genetic factors and biochemical adverse events. The World Health Organization (WHO) reports that there are currently over 1.2 billion people globally overweight and obese, and that in the United States, about 65% of the adult population is overweight. It is defined as chronic disease. Obesity is not only a matter of appearance, but is also reported to cause serious adult diseases such as diabetes, arteriosclerosis, cardiovascular diseases, hypertension and hyperlipidemia along with weight gain.

Recently, many researches for the development of obesity treatments are underway, and among them, a pancreatic lipase inhibitor is drawing attention. Pancreatic lipase acts as an important enzyme that breaks down triglycerides into 2-monoacylglycerols and fatty acids. A representative lipase inhibitor is tetrahydrolipstatin (Orlistat), a derivative of lipstatin derived from Streptomyces toxitricini , which is known to have the highest efficacy to inhibit about 30% of the fat consumed. It is currently on the market. However, despite these effects, orlistat is known to have side effects such as gastrointestinal disorders, hypersensitivity, bile secretion disorders, and fat-soluble vitamin absorption inhibition. Therefore, a recent research for the development of lipase inhibitors from natural materials with no side effects has been conducted.

Therefore, in the present invention, pogongyoung, ginger and gold were selected from natural herbal materials to determine their lipase inhibitory effect.

Pogong-young is a dried herb of Taraxacum platycarpum H. Dahlstedt or a plant of the same kind. Ocular hyperemia, acute hepatitis, jaundice, fever and urine due to symptoms, pharmacological action, immune function, bile secretion, hepatic function protection, diuretic effect has been reported.

Ginger (生 신선한) is a fresh root stem of ginger (Zingiber officinale Roscoe), which treats chills, fever, headache, vomiting, seawater and sputum caused by external colds, and is effective for abdominal diarrhea and stomachache caused by food poisoning. In addition, pharmacological action has been reported to promote gastric juice secretion, digestion, cardiac excitement, blood circulation, fungi.

Golden (黄 芩) is a perennial herb of the dicotyledon and moss on the moss. It is cultivated as a medicinal plant.

With respect to the herbal medicines pogongyoung, ginger and gold, Republic of Korea Patent No. 10-0479665 "Antioxidant enhanced cosmetic composition obtained by inoculating and fermenting effective microorganisms in brown rice, green tea, Pogongyoung", Republic of Korea Patent No. 10- 0088494 “Extracting and Purifying Extract with High Antimicrobial Activity from Pogongyoung” Republic of Korea Patent Application No. 10-2008-0076826 “External Skin Compositions Containing Pogongyoung, Horse Riding, Powder, or Gobon Extract”, Korean Patent Application No. 10-2005 -0042614 "Composition for the treatment and prevention of acute hepatitis containing pogongyoung extract", Korean Patent Application No. 10-2006-0138233 "Medicine and health food for the treatment and prevention of neurokinin mediated disease containing pogongyoung extract" and Korean Patent Application No. 10-2003-0011447 discloses a composition for treating and preventing liver disease containing pogongyeong extract

In addition, the Republic of Korea Patent Application No. 10-2009-7004765 No. "improved anti-inflammatory herbal composition and its use method" includes the extract extracted from the supercritical carbon dioxide extract of ginger with alcohol, turmeric, oregano, and ginger supercritical carbon dioxide A composition containing an extract and a hydroalcoholic extract of Holy Basil, Turmeric, Golden, Rosemary, Green Tea, Capsicum is disclosed. However, no anti-obesity studies using pogongyoung, ginger and gold have been conducted so far.

As described above, the present inventors conducted a thorough study by selecting pogongyoung, ginger, and gold to develop lipase inhibitors from natural materials, and confirmed that the mixtures thereof had excellent lipase inhibitory effects and completed the present invention. Was done.

It is therefore an object of the present invention to provide a pharmaceutical composition for the prevention and treatment of obesity, hyperlipidemia and fatty liver containing ginger, golden and pogongyoung.

In addition, another object of the present invention is to provide a dietary supplement for preventing and improving obesity, hyperlipidemia and fatty liver containing ginger, golden and pogongyoung.

An object of the present invention as described above is to prepare an extract or fraction of ginger, golden and pogongyoung, and to measure the inhibitory activity of lipolytic enzymes in consideration of the efficacy of each extract, the biological properties of the constituent compounds, etc. While establishing an optimal compounding ratio to maximize, a mixture of ginger, golden and pogongyoung effectively suppress weight gain due to high fat diet, significantly inhibit the size of fat cells can be useful in the treatment of obesity, It is effective in improving and treating hyperlipidemia caused by obesity by reducing lipids, and by reducing total cholesterol, effectively improving serum lipid levels, reducing hepatic lipid levels, and inhibiting the deposition of fat globules in liver tissues to improve fatty liver. Achieved by identifying the contribution.

The present invention provides a pharmaceutical composition for the prevention and treatment of obesity, containing ginger, golden and pogongyoung as an active ingredient.

In another aspect, the present invention provides a pharmaceutical composition for preventing and treating hyperlipidemia containing ginger, gold and pogongyoung as active ingredients.

In another aspect, the present invention provides a pharmaceutical composition for preventing and treating fatty liver containing ginger, golden and pogongyoung as an active ingredient.

In another aspect, the present invention provides a dietary supplement for preventing and improving obesity containing ginger, golden and pogongyoung as an active ingredient.

In another aspect, the present invention provides a health functional food composition for preventing and improving hyperlipidemia containing ginger, golden and pogongyoung as an active ingredient.

In another aspect, the present invention provides a health functional food composition for preventing and improving fatty liver containing ginger, golden and pogongyoung as an active ingredient.

In the present invention, the term "active ingredient" refers to a substance or a group of substances (including medicinal herbs whose pharmacologically active ingredients are not known) which are expected to express directly or indirectly the efficacy effect of the medicine by inherent pharmacological action. It means containing a main component as.

In the present invention, "health functional food" means a food manufactured and processed using raw materials or ingredients having functional properties useful for the human body according to the Health Functional Food Act No. 6767, and "functional" means a human body It means the ingestion for the purpose of obtaining a useful effect in health use such as nutrient control or physiological action on the structure and function of.

Pharmaceutical compositions comprising a mixture of ginger, gold and pogongyeong of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Pharmaceutical compositions comprising a mixture of ginger, golden and pogongyoung of the present invention, respectively, oral formulations, external preparations, suppositories, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like according to conventional methods It can be formulated and used in the form of sterile injectable solutions.

Carriers, excipients and diluents which may be included in pharmaceutical compositions comprising a mixture of ginger, gold and pogongyeong of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia Rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil Can be. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations include at least one excipient such as starch, calcium carbonate, sucrose in the extract. Or lactose, gelatin, or the like is mixed. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Preferred dosages of the mixture of ginger, gold and pogongyoung according to the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the mixture of ginger, golden and pogongyoung of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably from 0.001 to 100 mg / kg. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

The mixture of ginger, gold and pogongyoung according to the present invention can be administered to mammals such as mice, mice, livestock, humans, etc. by various routes. All modes of administration can be expected, for example, oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injections and directly to the skin.

In addition, the mixture of ginger, golden and pogongyoung according to the present invention can be added to food or beverage for the purpose of preventing and improving obesity, hyperlipidemia and fatty liver. Foods to which a mixture of ginger, golden and pogongyoung can be added include, for example, various foods, beverages, gums, teas, vitamin complexes, health functional foods, powders, granules, tablets, capsules or beverages. At this time, the amount of the mixture of ginger, golden and pogongyeong in the food or beverage can be added to 0.01 to 15% by weight of the total food weight, the health beverage composition is 0.02 to 5 g, preferably 0.3 to based on 100 ml It can be added at a rate of 1 g.

The food composition of the present invention is not particularly limited to other ingredients except for containing a mixture of ginger, golden and pogongyeong as essential ingredients in the ratios indicated, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. Can be. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (tauumatin, stevia extract (e.g., Rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of said natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

In addition to the above, the mixture of ginger, gold and pogongyoung of the present invention is a variety of nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, such as flavoring agents, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and Salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. In addition, the mixture of ginger, golden and pogongyoung of the present invention may contain pulp for the production of natural fruit juice and fruit juice beverage and vegetable beverage. These components can be used independently or in combination. The proportion of such additives is not so critical, but the mixture of ginger, gold and pogongyeong of the present invention is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight.

The mixture of pogongyoung, ginger and gold according to the present invention has excellent inhibitory activity of lipolytic enzymes, effectively inhibits weight gain due to high fat diet, significantly inhibits the size of fat cells, and can be useful for treating obesity. It is effective in improving and treating hyperlipidemia caused by obesity by reducing blood lipids, and by reducing total cholesterol, effectively improving serum lipid levels, reducing liver tissue lipid levels, and inhibiting the deposition of fat globules in liver tissue. It can help to improve fatty liver.

1 is a view schematically showing an experimental process according to the present invention.
Figure 2 shows the effect of herbal mixture supplementation on body weight in experimental diet rats for 5 weeks.
Figure 3 shows the effect of herbal mixture supplementation on serum cryglyceride concentrations in high fat diet-fed rats for 5 weeks.
FIG. 4 is a diagram showing the effect of herbal mixture supplementation on total cholesterol, HDL-cholesterol and LDL-cholesterol concentrations in high fat diet rats for 5 weeks.
FIG. 5 shows the effect of herbal mixture supplementation on hepatic lipid concentration in high fat diet fed rats for 5 weeks.
FIG. 6 shows the effect of herbal mixture supplementation on adipocyte morphology in high fat diet fed rats for 5 weeks.
FIG. 7 is a diagram showing the effect of herbal mixture supplementation on liver tissue morphology in high fat diet fed rats for 5 weeks.
Mean ± SE. ^abc differs significantly between groups ( p <0.05)
Normal control: normal diet
High-fat diet: high fat diet
CLA group: HFD diet + 2% CLA
Herb mixture: HF diet + 2% herbal mixture

Hereinafter, preferred embodiments of the present invention will be described in more detail with reference to Examples. However, the scope of the present invention is not limited to these examples.

Example 1: Preparation of herbal extracts for measuring lipase inhibitory activity

Each of these extracts was prepared to measure the lipase inhibitory activity of the pogongyoung, gold and ginger mixtures in this example. Poongongyoung, gold, ginger used as a sample was purchased from the 2009 Omni Hub, used as shown in Table 1.

TABLE 1

1.0 kg of each herbal medicine was finely pulverized with a grinder, and extracted three times for 3 days with 4 L of 80% methyl alcohol (MeOH), followed by polarization using an organic solvent. That is, 80% methanol extract was concentrated under reduced pressure to remove methanol, and the extracted sample suspended in water was first extracted with n -hexane, a low polar solvent, and the water layer was again extracted with ethyl acetate (EtOAc) and n -butyl alcohol ( n- BuOH). Each fraction was extracted three times sequentially.

Experimental Example 1 Measurement of Inhibitory Activity of Pancreatic Lipase

In this experimental example, the inhibitory activity of the lipase for each herbal extract prepared in Example 1 was measured using Kim's method. Enzyme buffer (10 mM MOPS, 1 mM EDTA, pH 6.8) and Tris buffer (100 mM Tris-HCl, 5 mM CaCl2, pH 7.0) were used before the experiment. Porcine pancreatic lipase was dissolved in enzyme buffer at 0.5 g / 200 ml and centrifuged at 4000 rpm for 5 minutes to use supernatant as enzyme. Substrate solution was prepared by dissolving 10 mM p -nitrophenyl butyrate in dimethylformamide. Sample reaction was performed by adding 169 μl of Tris buffer solution, 20 μl of herbal extract sample and 6 μl of enzyme into a 96 well plate, and reacting at 37 ° C. for 15 minutes. Then, 5 μl of substrate solution was added to make 200 μl. The absorbance was measured at 405 nm using an ELISA meter. Positive control experiments were carried out in the same way using orlistat (Genical).

As a result, the poongyoung 80% ethanol extract showed an inhibitory effect of 35.8% at the concentration of 31.3 ug / mL, and then the golden, ginger in the order of efficacy. The activity of each fraction obtained through the liquid-liquid fraction using organic solvents ( n -Hexane, CHCl ₃ , EtOAc, n -BuOH, etc.) for each crude drug showed strong activity in the EtOAc layer. Efficacy was equal to that of Xenical, an obesity treatment, and each fraction of ginger showed 99.7% inhibition at the concentration of 31.3 ug / mL of the EtOAc fraction, and 82.3% at the concentration of 31.3 ug / mL of the EtOAc fraction, too. Inhibitory activity was shown.

On the other hand, the results of measuring the lipase inhibitory activity against the herbal extract mixture is shown in Table 2,

TABLE 2

¹ positive control

² Each sample uses 80% EtOH extract.

³ pogongyoung: ginger: golden ratio.

As shown in Table 2, the mixing ratio of poongyoung, ginger, and gold is 1: 1: 1, 3: 1: 1, 5: 1: 1, 10: 1: 1, 1: 0.5: 1, 5: 0.5 In the case of 1: 1 and 10: 0.5: 1, all of the experimental concentrations of 31.3mg / ml resulted in the inhibition of lipase activity by more than 70%. Among them, it showed the highest efficacy at the ratio of 5: 0.5: 1, which was found to be nearly equivalent to 85.0% of inhibitory concentration at the experimental concentration of 31.3mg / ml of Zenical, a commercial obesity drug. The optimal compounding ratio that can inhibit fat absorption was found to be a ratio of 5: 0.5: 1 (pogongyoung: ginger: golden).

Example 2 Preparation of Herbal Extracts for In vivo Efficacy Evaluation

Each of these extracts was prepared to evaluate the in vivo efficacy of the pogongyoung, golden and ginger mixtures in this example.

First, three times (v / v) of 8% EtOH was added to ginger, pogongyoung and gold, and immersion extraction was repeated three times at room temperature for 24 hours, followed by filtration with filter paper (Whatman No. 1), and then Each extract was concentrated under a rotary vacuum evaporator at 40 ° C. or lower. The obtained extract sample was lyophilized and powdered, mixed at a constant ratio and used as a sample for the following experiment.

Experimental Example 2 In vivo efficacy evaluation of herbal extracts

In this experimental example, the normal, obese control group (high fat diet group) and positive control group (CLA) were tested to verify the anti-obesity effect of the herbal mixture mixed with ginger, poongongyoung and gold on the experimental animals induced by obesity. Group) and herbal mixtures were compared and verified. Experimental animals were purchased from Sprague-Dawley (5 weeks old, 145 ± 5 g, 28) through Orient Co., Ltd. After one week of adaptation to a lab-chow pellet, the other groups except the normal control group ingested the high-fat diet freely for 5 weeks to induce obesity, and then the high-fat diet group, CLA group and herbal medicine as shown in FIG. The experimental diet was fed for 5 weeks, divided into three groups of mixtures (7 per group). The experimental diet was based on the AIN-76 diet (Teklad, USA), the high-fat diet fed a high-fat diet (20% fat), and the CLA and herbal mixtures added 2% of the test substance to the high-fat diet. One diet was fed (Table 3). The CLA group added 2% CLA instead of 2% corn oil, and the herbal mixture added 2% sucrose and the 2% herbal mixture containing poongyoung, ginger, and gold in a proportion. Experimental diet and drinking water were to be freely ingested, and the diet was refrigerated at 4 ℃ during the breeding period. The animals were kept in individual cages, and the breeding environment of the animal laboratory was constant at constant temperature (25 ± 2 ° C), humidity (60 ± 5%), and dark-light cycles at 12-hour intervals. The condition was maintained. Body weight and dietary intake were measured at regular intervals over two days during the entire experimental period. The feed efficiency ratio (FER) was calculated by dividing the weight gain in the experimental period by the amount of food consumed during the same period.

TABLE 3

Experimental diet composition (g / kg diet)

¹⁾ AIN mineral mixture (g / kg): calcium lactate 620.0, sodium chloride 74.0, potassium phosphate dibasic 220.0, potassium sulfate 52.0, magnesium oxide 23.0, manganese carbonate 3.3, iron citrate 6.0, zinc carbonate 1.0, copper carbonate 0.2, iodide 1,000 g of finely divided potassium 0.01, sodium selenite 0.01, potassium chromium sulfate 0.5.

²⁾ AIN Vitamin Mixture (mg / kg): Thiamine-HCl 600, Riboflavin ± 600, Pyridoxine-HCl 700, Nicotinic Acid 3,000, D-Calcium Pantothenate 1,600, Folic Acid 200, D-Biotin 20, Vitamin B12 2.5, Vitamin 1,000 g of A400,000 IU, vitamin D3 100,000 IU, vitamin E 7,500 IU, and vitamin K 75.

After the animal was bred for 12 hours (PM 9:00-AM 9:00) before the sacrifice, the animals were inhaled by primary inhalation with ether, and fasted blood was collected from the inferior vena cava. It was. Serum was separated by centrifugation at 3,000 × g, 4 ° C. for 15 minutes, and the collected blood was stored at −70 ° C. until sample analysis. Organ tissues (liver, heart, kidney) of each experimental animal were rinsed several times in PBS (phosphate buffered saline) solution and weighed by removing surface moisture. The liver was collected separately for enzyme activity measurement and lipid quantification, quenched in liquid nitrogen and stored at -70 ° C until sample analysis.

All the results of the experiment were calculated using a computer statistical program (SPSS package program). Significance test for mean difference between each group was performed by one-way ANOVA (analysis of variance) and post-test was performed at p <0.05 level by the Duncan's multiple range test. Are expressed as mean ± standard error.

Experimental Example 2-1: Dietary Intake and Long-term Weight

Dietary intake and organ weights during the experimental period are shown in the following Tables 4 and 5. There was no significant difference in dietary intake in all groups during the experimental period. Therefore, it was considered that dietary intake did not affect the increase or decrease of body weight. The weight gain and dietary efficiency of the high-fat diet group and the CLA group ingesting the high-fat diet were the highest, and the herbal mixture group showed the least weight gain and the diet efficiency was also low. The dietary efficiency is obtained by dividing the weight gained during the experimental period by the dietary intake. The low dietary efficiency of the herbal mixture group, despite the same dietary intake, means that the weight gain by dietary intake is low. Therefore, herbal mixtures are thought to be controlled by metabolic causes without being affected by dietary intake. On the other hand, long-term intake of high-fat diet caused non-alcoholic fatty liver, and in this experiment, liver weight of high-fat diet group, CLA group, and herbal medicine mixture group significantly increased compared to normal control group, and added CLA or herbal mixture There was no significant effect by.

TABLE 4

Effect of herbal mixture supplementation on weight gain, dietary intake and dietary efficiency in rats fed experimental diet for 5 weeks

Mean ± SE. ^ab is significantly different between groups ( p <0.05)

Normal control: normal diet

High Fat Diet: High Fat Diet (HFD)

CLA group: HFD + 2% CLA

Herbal mixture: HFD + 2% herbal mixture

¹⁾ FER (food efficiency ratio) = weight gain / food intake

NS: not significant

TABLE 5

Effect of Herbal Mix Supplementation on Organ Weight in Rats Fed Experimental Diet for 5 Weeks

Mean ± SE. ^abc differs significantly between groups ( p <0.05)

Normal control: normal diet

High Fat Diet: High Fat Diet (HFD)

CLA group: HFD + 2% CLA

Herbal mixture: HFD + 2% herbal mixture

BW: weight

Experimental Example 2-2: weight change

During the experiment, the weights of all groups increased steadily (FIG. 2). After induction of obesity for 5 weeks, the weight of the first week of dietary diet was significantly increased in all the groups fed the high-fat diet compared to the normal control group. At 2 and 3 weeks of feeding, the weights of the CLA and herbal mixtures were significantly lower than those of the high-fat diet. At the end of the 5 weeks, the CLA group tended to lose weight than the high-fat diet group, and the herbal mix group significantly lost weight. In addition, the herbal mixture group showed the most modest weight gain during the five weeks of high fat diet, and the dietary supplement of ginger, poongongyoung and golden mixture was found to effectively suppress the weight gain by the high fat diet.

Experimental Example 2-3: Total Cholesterol, HDL-Cholesterol and LDL-Cholesterol Assay

Serum total cholesterol was measured using a test solution (Asanpharm Co., Korea) applying the enzyme method of Allain et al. (1974). Serum cholesterol is present in both CE and free cholesterol, so in order to quantify both, CE was converted to fatty acids and free cholesterol by cholesterol cholesterol esterase. The free cholesterol thus converted is converted into H ₂ O ₂ and Δ ⁴ -cholesterone by cholesterol oxidase, among which H ₂ O ₂ is converted to peroxidase, phenol, 4-amino After mixing with anti-thrift and red color, the absorbance at 500 nm was measured and quantified in comparison with cholesterol standard solution (300 mg / dL).

HDL-cholesterol and LDL-cholesterol were measured using an automated serum analyzer (Thermo Electron, USA).

Serum total cholesterol concentration was significantly increased in the three groups fed the high fat diet than the normal control group. In the high fat diet group, the total cholesterol concentration was increased 1.7 times than the normal control group, and the CLA group and the herbal medicine mixture decreased by 13% and 8%, respectively, by the addition of CLA and the herbal mixture. LDL-cholesterol was significantly decreased in the CLA group compared to the high-fat diet group, but there was no significant difference by supplementing herbal mixture. In addition, serum HDL-cholesterol concentration was lower than that of the normal control group in the high-fat diet group, but it was not decreased by supplementation of CLA and herbal mixture, and the herbal mixture reduced total cholesterol without decreasing HDL-cholesterol. Therefore, the herbal mixture was found to effectively improve serum lipid levels by reducing total cholesterol while maintaining HDL-cholesterol at normal levels.

Experimental Example 2-4: Triglyceride Analysis

Serum triglycerides were measured using a reagent for measuring triglycerides (Asanpharm Co., Korea) according to the principle of color development using the enzyme method of McGowan et al. (1983). Serum triglycerides are broken down into glycerol and fatty acids by lipoprotein lipase (LPL). Among them, glycerol forms L-α-glycerophosphate by the action of ATP and glycerol kinase (GK), which reacts with O ₂ and GPO (glycerophosphooxidase) to generate H ₂ O ₂ . Peroxidase and 4-amino-antipyrine were treated to develop red color, and then absorbance was measured at 550 nm and quantified by comparison with a glycerol standard curve.

Induction of obesity by high-fat diet leads to a sharp increase in blood lipid levels. In this experiment, the triglyceride concentration of the high-fat diet fed high-fat diet was 157.2 mg / dL, which was about 2 times higher than the normal control 68 mg / dL. Meanwhile, the serum triglyceride concentrations of CLA and herbal mixtures with CLA and herbal mixtures added to the high-fat diet were 102.7 mg / dL and 107.3 mg / dL, respectively. 34.6% and 31.7%, respectively, were significantly decreased. In particular, the serum triglyceride concentration in the herbal mixture group was similar to that of CLA, an anti-obesity functional ingredient. Ginger, pogongyoung and golden mixtures were found to be very effective in improving and treating hyperlipidemia due to obesity.

Experimental Example 2-5: Measurement of liver tissue neutral lipid and cholesterol content

Hepatic lipids were extracted by Folch et al. (1957), and hepatic triglyceride and cholesterol content were determined by Sale et al. (1983). 0.5 g of liver tissue was chopped and homogenized with 5 mL chloroform: methanol (2: 1) solution to extract tissue lipids. The extract was filtered with filter paper (Whatman No. 2), dried over nitrogen gas and dissolved in 1 mL of the same extractant. Among them, 100 μL of neutral lipids and total cholesterol were taken, dried with nitrogen gas, dissolved in 5 mL of ethanol, and liver lipids were quantified by the same enzyme reaction as plasma lipid quantification method.

Non-alcoholic fatty liver is a case where fatty acids are accumulated in hepatocytes in the form of triglycerides, not by alcohol or hepatitis virus, and are caused by obesity, diabetes, hyperlipidemia, and insulin resistance and metabolic syndrome. The mechanism of fatty liver development is not clear, but fatty liver accumulates fat in hepatocytes due to insulin resistance and hepatic fat metabolism disorder.The next step is imbalance between pro-inflammatory and anti-inflammatory cytokines, fatty peroxidation and activity in liver. It has been suggested that fatty hepatitis is caused by oxygen species. Therefore, high-fat diet ingestion induced hyperlipidemia, obesity, and induces non-alcoholic fatty liver. In this experiment, we measured the effect of reducing the liver tissue lipid concentration of the herbal mixture in experimental animals induced by fatty liver. As a result, the hepatic triglyceride concentration (130.17 ± 8.01 mg / g tissue) of the high fat diet group induced by fatty liver by ingesting high fat diet was more than four times higher than the normal control group (29.66 ± 1.03 mg / g tissue). Hepatic triglyceride concentrations of 2% CLA and herbal mixtures were significantly lower than those of high fat diet. Therefore, dietary supplementation of ginger, pogongyoung and gold mixtures were found to significantly reduce liver lipid levels and contribute to fatty liver improvement.

Experimental Example 2-6: Liver and Adipose Tissue Morphological Analysis

Morphological observations of liver tissue and epididymal white fat cells were performed as follows. Some of the liver tissue and epididymal white adipose tissue extracted at animal sacrifice were fixed in 10% formaldehyde solution for 24 hours and then exchanged twice with the same solution. Then, dehydrated with doubling ethanol and embedded in paraffin, which was cut to 4 μm thickness, stained with hematoxylin-eosin (H-E), and observed under a 200-fold magnification of liver tissue and epididymal white fat cells under an optical microscope. Adipocyte size was determined by Leica Application Suite. It was measured using Ver 2.8.1 (Leica). Liver tissue is grade 1 (1-25%), grade 2 (25-50%), grade 3 (50-75%), grade 4 (75-100%) based on the percentage of fat spheres (fatty spheres / total liver). Was evaluated. In addition, the proportion of fatitis lesions was evaluated as 1st grade (1-25%), 2nd grade (25-50%), 3rd grade (50-75%), and 4th grade (75-100%). The results were calculated by adding the hepatic macrovesicular steatosis grade.

In fatty liver induced by obesity, micro fat cells accumulate in the hepatoplasm and these micro fat cells progress to macrovesicular steatosis and large fat cells appear in tissues. In this experiment, fat accumulation was observed in the liver tissues of the high fat diet group, CLA group, and the herbal medicine mixture group, which were induced by high fat diet. In addition, four grades were obtained according to the degree of fat globule deposition in the liver, and the grades were calculated according to the proportion of fatitis lesions, and the results (hepatic macrovesicular steatosisi grade) were calculated. The CLA and herbal mixtures were reduced to half the normal controls. Therefore, herbal mixtures have been shown to contribute to fatty liver improvement by inhibiting the deposition of fat globules in liver tissue.

In addition, if obesity is induced due to high fat diet, fat cell number and size will increase significantly. Therefore, the effects of five-week experimental diet on the morphological changes of the epididymal adipose tissue after inducing obesity by the high fat diet were as follows. The high-fat diet group fed high-fat diet for 10 weeks had twice the size of fat cells compared to the normal control group (A, C). On the other hand, the fat cell size was significantly decreased in the CLA group and the herbal mixture group in which obesity was induced by the high-fat diet for 5 weeks and then fed the diet containing CLA and the herbal mixture. The larger the adipocyte size, the smaller the number of cells observed in the same field of view. The high-fat diet group showed the lowest number of adipocytes observed in the same field of view in contrast to the adipocyte size (B). On the other hand, the CLA group and the herbal mixture group had a smaller fat cell size than the high-fat diet group, which significantly increased the number of cells observed in one field of view. Through this, the herbal mixture used in this experiment was observed to significantly inhibit the increase of fat cell size, it could be confirmed that it can be useful for the treatment of obesity caused by high fat diet.

Claims (6)

Pharmaceutical composition for the prevention and treatment of obesity containing ginger, gold and pogongyoung as active ingredients. Pharmaceutical composition for the prevention and treatment of hyperlipidemia containing ginger, golden and pogongyoung as active ingredients. Pharmaceutical composition for preventing and treating fatty liver containing ginger, golden and pogongyoung as active ingredients. Health functional food composition for the prevention and improvement of obesity containing ginger, golden and pogongyoung as an active ingredient. Health functional food composition for preventing and improving hyperlipidemia containing ginger, golden and pogongyoung as an active ingredient. Health functional food composition for preventing and improving fatty liver containing ginger, golden and pogongyoung as an active ingredient.

Images