KR20130017305A - A composition comprising an extract of fermented or non-fermented lonicerae flos and citri reticulatae pericarpium for treating or preventing obesity - Google Patents

Abstract

PURPOSE: A composition containing a fermented or an unfermented extract of Lonicerae Flos and Citri Reticulatae Pericarpium is provided to suppress fat differentiation in 3T3-L1 cells and to be used in a medicine or a health functional food for preventing and treating obesity. CONSTITUTION: A pharmaceutical composition for preventing and treating obesity contains a fermented or an unfermented extract of Lonicerae Flos and Citri Reticulatae Pericarpium as an active ingredient. The extract is prepared using water, low carbon number alcohol of C1-C4, or a mixture thereof. A health functional food for preventing and treating obesity contains the extract as an active ingredient.

Description

A composition comprising an extract of fermented or non-fermented Lonicerae Flos and Citri Reticulatae Pericarpium for treating or preventing obesity}

The present invention relates to a health functional food for the prevention and treatment of obesity containing fermented or unfermented gold and silver dermis mixed herbal extract as an active ingredient.

Document 1 Van der Ploeg LH. Related Obesity: an epidemic in need of therapeutics. Curr Opin Chem Biol. 4 (4) , pp.452-460, 2004

[2] Kim DM, et al., Prevalence of obesity in Korea. Obes Rev. 6 , pp. 117-121, 2005

[Reference 3] Daily JW, Cha YS. Macronutrient intake and obesity. J Food Sci Nutr. 5 (1) , pp. 58-64, 2000

[Reference 4] 張介賓.張氏 類 經. Seongbosa, Seoul. p.586, 1982

[Document 5] Eun-Seon Kim et al 2, Obesity Therapeutics, In-depth Information Analysis Report, Korea Institute of Science and Technology Information, pp.1-111, 2002

[Reference 6] National Institute of Oriental Medicine Joint Textbook Compilation Committee, Herbology, Younglim History, pp.242-244, 2006

[Reference 7] National Institute of Oriental Medicine, Joint Textbook Editing Committee, Herbology, Younglimsa, pp.466-469, 2006

[Reference 8] Park, Jung-Yeon, and San-Sa-eun, The Effect of Tea on Body Serum Lipids and Oxidative Stress in Overweight Women, Master's Thesis, Myongji University, 2009

[9] Choi, Jong-Hwan et al 7, Effects of dermal combination and exercise on physique, body fat and blood lipid in obese women, Journal of Pharmacognosy, 33 (1) , pp. 57-63, 2002

[Reference 10] Park Chi-deok, Aureobasidium Study on biotransformation and anti-obesity effects of flavonoid dermis using pullulans, PhD thesis, Keimyung University, 2011

[11] Murase T. et, al, Nootkatone, a characteristic constituent of grapefruit, stimulates energy metabolism and prevents diet-induced obesity by activating AMPK, Am J Physiol Endocrinol Metab, 299 (2) , pp. E266-75, 2010 )

12. Wolfe K. et al., Antioxidant activity of apple peels. J. Agric. Food. Chem. 51 , pp. 609-614, 2003

[Reference 13] Lin. J. et al., Green Tea Polyphenol Epigallocatechin Gallate Inhibits Adipogenesis and Induces Apoptosis in 3T3-L1 Adipocytes, Obesity Research, 13 (6) , pp.982-990, 2005

Lee JS et al., Supplementation of whole persimmon leaf improves lipid profiles and suppresses body weight gain in rat fed high-fat diet, Food and Chemical Toxicology, 44 (11) , pp. 1875-1883, 2006

Huang B. et, al, Cinnamaldehyde prevents adipocyte differentiation and adipogenesis via regulation of peroxisome proliferator-activated receptor-γ (PPARγ) and AMP-activated protein kinase (AMPK) pathways, J Agric Food Chem, Apr 27; 59; (8) , pp. 3666-73, 2011

[16] Allain C. et al., Enzymatic determination of total serum cholesterol, Clin. Chem. 20 (4) , pp.470-475, 1974

17. Van Handel E. et al, Micromethod for the direct determination of serum triglycerides, J. Lab. Clin. Med. 50 (1) , pp.152-157, 1957

[Document 18] Warnick GR et al., Dextran sulfate-Mg 2 + precipitation procedure for quantitationof high density lipoprotein cholesterol, Clin. Chem. 20 (6) , pp. 1397-1388, 1982

Giusti G. et al., A comparative study of some spectrophotometric and colorimetric procedures for the determination of serum glutamic-oxaloacetic and glutamic-pyruvic transaminase in hepatic diseases. Enzymol. Biol. Clin (Basel), 10 (1) , pp. 17-38, 1969

DiGirolamo M. et al., Qualitative regional differences in adipose tissue growth and cellularity in male Wistar rats fed ad libitum. Am. J. Physiol. 274 , pp.R1460-1467, 1998

Obesity is a phenomenon caused by an imbalance between the intake and consumption of energy, which means an excess of fat in the body. Although it is not known yet, because it is caused by various causes, it is generally recognized to be caused by excessive calorie intake, endocrine disorders, lack of exercise, and genetic factors. It is also commonly referred to as lifestyle disease because it is closely related to lifestyle. Since 2000, the number of people with metabolic syndrome, which is susceptible to chronic diseases such as heart disease and diabetes, has increased 65% in recent five years. As a result, various problems such as suffering and poor quality of life of the patient as well as excessive medical expenses and reduced productivity are socially occurring.

The World Health Organization estimates that as of 2005, one billion people in the world are obese, and over 30% of all deaths are related to metabolic diseases, including obesity. It is estimated that metabolic diseases will increase by about 50% to 1.5 billion people.

Obesity refers to a condition in which the body's fat is excessively accumulated to damage health, and can be defined as obesity when there is an increase of 20% or more of the individual's standard weight. Specifically, the weight of fat accounts for more than 25% of body fat in men and more than 30% in women (Van der Ploeg LH.Related Obesity: an epidemic in need of therapeutics.Curr Opin Chem Biol 4 (4) , pp. 452-460, 2004). The number of people who are overweight or obese is increasing all over the world, but the incidence of obesity is increasing in Korea recently (Kim DM, et al., Prevalence of obesity in Korea.Obes Rev. 6 , pp.117-121 , 2005). Obesity is associated with the prevalence of various degenerative diseases, such as diabetes, hypertension, cardiovascular disease, joint disease, lung disease, and some cancers, because excess energy is converted to fat due to an imbalance between energy intake and consumption. The financial burden and loss of life incurred are enormous (Daily JW, Cha YS. Macronutrient intake and obesity. J Food Sci Nutr. 5 (1) , pp.58-64, 2000). In the case of Asians in particular, obesity management is more important because the body mass index is at least abdominal obesity compared with Westerners, and the sensitivity of arterial diseases such as hypertension, diabetes, and hyperlipidemia is high. In oriental medicine, the causes of obesity are regarded as 膏粱 珍味, 氣虛, 濕 滯, etc., and as a treatment, 약물 水 化濕, 化痰, 熱 通腑, 活血 祛瘀 and drug therapy (張介賓. 張 서울. Seoul, Seongbosa, p.586, 1982). Obesity treatments include appetite suppressants, liposuction inhibitors, energy metabolism accelerators, hormonal preparations, and surgical procedures such as gastrectomy and liposuction. Symptoms, etc. (Van der Ploeg LH.Related Obesity: an epidemic in need of therapeutics.Cur Opin Chem Biol. 4 (4) , pp.452-460, 2004; Report, Korea Institute of Science and Technology Information, pp.1-111, 2002). Currently, the medical community recognizes the appetite suppressant sibutramine and the fat absorption inhibitor olistat as obesity drugs that can be used for a long time, but there are still questions about side effects, so diet, exercise and behavior therapy are used to safely lose weight. Including a multifaceted approach is recommended.

Lonicerae Flos is a perennial herbaceous vine that belongs to the Caprifoliaceae family. It is harvested before flowering in summer and dried in shade. Tannin, inositol, sterol, chlorogenic acid and isochlogenic acid have been reported as ingredients, and flavonoids include luteolin, apigenin and luteolin-7-O-rhamnoglucoside. Contains lonicerin, inositol, essential oils and quercetin. It is sweet and weak in coldness and detoxification, so it is an important medicine to cure heat venom (Jungkuk University of Medicine, College of Medicine, Herbal Medicine, Younglimsa, pp.242-244 , 2006).

The dermis (Citri Reticullatae Pericarpium) is a dry-married fruit peel of the mature fruit of the evergreen subfamily, Tangerine, and the same genus, which belongs to the Unhyang family. It is harvested in November and dried in the sun. Its components include hesperidin, naringin poncirin, nobilein, neoesprin, nobileting, neohesprindin and tanderetin as flavonoids. The taste is spicy and bitter, the medicinal properties are warm, fragrant, and mainly enter into the rain and menopause, which is effective. The spicy taste is divergent and hangs, and the taste is bitter and warm. By the way, if you let the siege acts, the spleen and the spleen will be healthy, and if the humidity of the spleen is removed, the cough will go away on its own. This product becomes an important medicine for selfishness, dryness, humping, and conversation. (College of Teaching Materials, National Institute of Oriental Medicine, Herbology, Younglimsa, pp.466-469, 2006 ).

Recently, interest in herbal medicine has been amplified in the Chinese medicine academy. Fermented Chinese medicine refers to herbal medicines fermented using microorganisms in the air or purely isolated microorganisms such as lactic acid bacteria, after boiling or boiling the traditional herbal medicines so that they can be utilized by microorganisms through traditional fermentation techniques. It is a kind of processing method that maximizes the body absorption rate and bioavailability of Chinese herbal medicines and improves the pharmacological function as well as the formulation improvement and preparation method of Chinese medicine, thereby creating new demand for Chinese medicine and producing new high value-added Chinese medicine products. It is meant to be able to develop. Currently, fermented herbal medicines with various preparations and forms are being developed. Especially, fermented red ginseng products and fermented herbal cosmetics are representative products.

However, none of the above documents teaches or discloses that the mixed herbal extract obtained by fermenting sterling silver and dermis can be used as a composition for preventing and treating obesity.

Therefore, the present inventors have conducted research to develop an effective therapeutic agent for obesity, inhibiting the differentiation of 3T3-L1, which is a fat cell, from gold and silver dermis fermented extracts, and reducing the body weight, body fat, and blood lipid levels in obesity-induced animals using high-fat diet. In addition to the reduction of leptin (leptin), adiponectin (adiponectin) resulted in a decrease in blood concentration, and the present invention was completed by confirming that the diameter of the epididymal fat cells.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention and treatment of obesity containing fermented or unfermented gold and silver dermis mixed herbal extract as an active ingredient.

The present invention also provides a health functional food for the prevention and improvement of obesity, containing fermented or unfermented sterling silver and dermis mixed herbal extracts as an active ingredient.

Mixed herbal extracts as defined herein include extracts soluble in water, preferably lower alcohols having 1 to 4 carbon atoms or mixed solvents thereof, preferably extracts soluble in water.

Preferred weight blending ratios of the mixed herbals as defined herein include blends in which the weight blending ratio (w / w) of gold to silver dermis is 1-5: 1, more preferably 1-3: 1 (w / w). It is characterized by that.

The fermentation extracts defined herein are sterilized from 10 to 70 minutes, preferably from 20 to 30 minutes, at 80 to 150 ° C. for samples of dried individual herbs with a water content of less than 20%, preferably less than 10%. Sterilization step; Sterilized samples can be fermented for Aspergillus oryzae, Aspergillus niger, Lactobacillus acidophilus Lactobacillus casei, and other food processing. A strain, preferably Aspergillus oryzae, is added in an amount of 1 to 30%, preferably 1 to 20% of the weight of the sample so that 10 to 50%, preferably 20 to 40% A second step of carrying out fermentation under conditions at a temperature of 10 to 50 ° C., preferably at 20 to 40 ° C., for 1 to 30 days, preferably 5 to 20 days, more preferably 8 to 12 days; A third step of drying the fermentation having completed the fermentation for 12 hours to 72 hours, preferably 24 hours to 52 hours at a temperature of 30 to 100 ℃, preferably 40 to 60 ℃; A fourth step of releasing the dried agglomerated particles to separate themselves and separating and selecting the fine particles; The selected fermented product is a process comprising a fifth step of performing a process of quenching for 5 minutes to 60 minutes, preferably 10 minutes to 30 minutes with far-infrared herb at 100 to 300 ℃, preferably 150 to 200 ℃ Obtainable.

Hereinafter, the present invention will be described in detail.

The mixed herbal extract of the present invention can be obtained as follows.

Fermented or unfermented mixed herbal extract of the present invention, first, the gold and silver or dermis individual herbal medicines, the water moisture content of less than 20%, preferably less than 10% of the sample dried at 80 to 150 ℃ 10 minutes to 70 minutes, preferably The sterilization step of undergoing a sterilization process for 20 to 30 minutes; Sterilized samples can be fermented for Aspergillus oryzae, Aspergillus niger, Lactobacillus acidophilus Lactobacillus casei, and other food processing. The strain is added in an amount of 1 to 30%, preferably 1 to 20%, of the sample weight, so that the temperature is 10 to 50 ° C., preferably 20 to 40 ° C. under conditions of 10 to 50%, preferably 20 to 40%, moisture. A second step of performing fermentation in 1 to 30 days, preferably 5 to 20 days, more preferably 8 to 12 days; A third step of drying the fermentation having completed the fermentation for 12 hours to 72 hours, preferably 24 hours to 52 hours at a temperature of 30 to 100 ℃, preferably 40 to 60 ℃; A fourth step of releasing the dried agglomerated particles to separate themselves and separating and selecting the fine particles; The selected fermented product is subjected to a fifth step of carrying out the process of sieving for 5 minutes to 60 minutes, preferably 10 minutes to 30 minutes with far-infrared herb at 100 to 300 ° C, preferably 150 to 200 ° C. Fermentation samples of can be obtained.

The fermentation sample obtained in the above step or the unfermented dry medicinal herb gold and silver dermis are washed, and the weight mixing ratio (w / w) of gold to silver dermis is 1-5: 1, more preferably 1-3: 1 (w / w) and 1 to 20 times (w / v) weight of the sample, preferably 1 to 10 times (w / v) weight of water, methanol, ethanol or a mixed solvent thereof, preferably 50 to water The extract obtained after performing hot water extraction, cold needle extraction or ultrasonic extraction, preferably hot water extraction at 120 ° C., preferably about 80-100 ° C. for 1 hour to 5 hours, preferably 2 hours to 4 hours, is filtered with a filter paper. The filtrate obtained after the filtration may be lyophilized, room temperature or hot air dried, preferably lyophilized, to obtain fermented or unfermented mixed herbal extracts composed of gold and silver, the dermis of the present invention.

The present invention provides a pharmaceutical composition for the treatment and prevention of obesity, containing the fermented or unfermented sterling silver and dermis herbal extract obtained by the manufacturing method and the manufacturing process as an active ingredient.

The gold and silver dermis herbal extracts obtained above reduce fat differentiation of adipocytes, and show weight loss, body fat reduction and blood lipid concentration in rats induced obesity using high fat diet.

The pharmaceutical composition for treating obesity of the present invention comprises 0.01 to 80% by weight of the extract, preferably 10 to 50% by weight, based on the total weight of the composition.

Pharmaceutical compositions comprising the extract of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Pharmaceutical dosage forms of the extracts of the present invention may be used in the form of their pharmaceutically acceptable salts, or may be used alone or in combination with other pharmaceutically active compounds, as well as in any suitable collection.

The pharmaceutical composition comprising the extract according to the present invention is formulated in the form of external preparations, suppositories, and sterile injectable solutions, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc. Can be used. Carriers, excipients and diluents that may be included in the composition comprising the extract include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, weights, binders, wetting agents, sealing agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, sucrose, and the like in the extracts and fractions. (sucrose), lactose (lactose), gelatin, etc. are mixed and prepared. 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 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. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol gelatin and the like can be used.

The preferred dosage of the pharmaceutical composition comprising the extract of the present invention depends 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 pharmaceutical composition comprising the extract of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably 0.001 to 100 mg / kg per day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

Fermented or unfermented sterling silver and dermis extracts of the present invention can be administered to mammals such as mice, mice, livestock, humans and the like by a variety of routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or Intracerebroventricular injection.

In addition, the present invention provides a health functional food for the prevention and improvement of obesity, containing fermented or unfermented sterling silver and dermis mixed herbal extracts having an excellent inhibitory effect on obesity as an active ingredient.

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

Functional food for the prevention and improvement of obesity of the present invention, the extract comprises 0.01 to 95% by weight, preferably 1 to 80% by weight relative to the total weight of the composition.

For the purpose of prevention and improvement of obesity, it is also possible to use a pharmaceutical dosage form such as a powder, a granule, a tablet, a capsule, a pill, a suspension, an emulsion or a syrup or a health functional food in the form of a tea bag, Processing is possible.

The present invention also provides a dietary supplement containing as a main ingredient a mixed herbal extract of fermented or non-fermented gold and silver and dermis exhibiting anti-obesity activity.

The health functional beverage composition of the present invention has no particular limitation on the other ingredients other than the above-mentioned extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. 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 natural flavors other than those described above, natural flavors (such as tau martin, stevia extract (e.g., rebaudioside A, glycyrrhizin)) and synthetic flavors (saccharin, aspartame, etc.) have. The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. The compositions of the present invention may also contain pulp for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

In addition, the extract of the present invention may be added to food or beverage for the purpose of preventing obesity. At this time, the amount of the extract in the food or beverage can be added in 0.01 to 15% by weight of the total food weight, the health beverage composition is added in a ratio of 0.02 to 5 g, preferably 0.3 to 1 g based on 100 ml Can be.

Fermented or unfermented mixed gold and silver extracts of the present invention are effective in inhibiting fat differentiation in mouse embryo fibroblast-derived adipocytes, 3T3-L1, and in obese rats induced by high fat diet. In addition to weight loss, body fat reduction and blood lipid concentrations, leptin and adiponectin reduce blood levels, which can be useful in pharmaceutical compositions, dietary supplements, and health supplements for the prevention and treatment of obesity. Can be.

1 is a diagram showing the effect of F-CL on MTT assay of 3T3-L1, a cell line of adipocytes,
2 is a diagram showing the effect of F-CL on Oil Red-O staining of 3T3-L1, a cell line of adipocytes,
3 is a diagram showing the effect of CL and F-CL on the effect of the herbal medicine extract on AMPK and ACC activity,
Figure 4 is a diagram showing the effect of F-CL on the weight of obese mice due to high-fat diet administration,
5 is a diagram showing the effect of F-CL on the liver weight of obese mice due to high-fat diet administration,
Figure 6 (ad) is a diagram showing the effect of F-CL on the body fat weight of obese mice due to high fat diet administration,
Figure 7 (af) is a diagram showing the effect of F-CL on the blood lipid concentration of obese mice due to high-fat diet administration,
8 is a diagram showing the effect of F-CL on the concentration of Adiponectin, Leptin in the blood of obese rats due to high-fat diet administration,
9 is a diagram showing the effect of F-CL on AMPK and ACC activity in the tissues of obese mice due to high-fat diet administration.

Hereinafter, the present invention will be described in detail with reference to the following examples and experimental examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the contents of the present invention are not limited by the following Examples, Reference Examples and Experimental Examples.

Example 1 Fermentation and Extract Preparation of Gold Coin and Dermis Herbal Medicine

1-1. Preparation of Gold Silver Fermentation

After preparing and selecting 15 kg of gold and silver coins (purchased Xinhuashan) dried with moisture content less than 10% and sterilizing with a sterilizer (CT-DAC 80 and Coretech) for 20 minutes at 120 ℃, fermented strain (Aspergillus oryzae) Fermentation was carried out in a fermentation apparatus (temperature controlled fermentation chamber, Izobio Co., Ltd.) for 10 days at a temperature of 30 ° C. under a condition of 30% of moisture, to obtain fermentation product. After fermentation is completed, the fermented product is dried at 57 ° C. for 48 hours, the agglomerated particles are lightly rubbed by hand to be separated by self, and the fine particles are separated and selected.

After quenching for 20 minutes with far-infrared herb at 180 ° C. to obtain 13 Kg of sterling silver fermentation was used below.

1-2. Preparation of Dermal Fermentation

Prepare and pulverize 18 kg of dried dermis (new product) with moisture content less than 10%, separate the particle size of each raw material to 0.2-0.5 mm, and sterilizer for 20 minutes at 120 ℃ (CT-DAC 80, Coretech) After the sterilization process, the fermented strain (Aspergillus oryzae) was added to 10% of the sample weight, and the fermentation apparatus (Izobio Co., Ltd.) The fermentation was carried out to obtain the fertilized fermented gold. After fermentation is completed, the fermented product is dried at 57 ° C. for 48 hours, the agglomerated particles are lightly rubbed by hand to be separated by self, and the fine particles are separated and selected.

Subsequently, the process was conducted for 20 minutes with far-infrared herb at 180 ° C. to obtain 15 Kg of dermal solid fermentation product, which was used below.

1-3. Preparation of Fermentation Blend Extract

After fermenting the gold and fermented by the above process, the dermis, unfermented gold and silver dermis 5 kg in a 3: 2 weight ratio, 10 times distilled water was added to the water and then raised to 95 ℃ and extracted for 12 hours. After the extraction, the precipitate was filtered using filter paper (Advantec, 71101903), and the filtrate was concentrated under reduced pressure in a rotary vacuum distillation apparatus (EYELA, A-1000s). The concentrate was left for 12 hours in a -70 ° C. deep freezer and dried for 72 hours in a freeze dryer for 2.1 kg (yield 42%) of fermented mixed extract powder and the fermentation process was not performed. 1.6 kg (yield 32% yield) of the non-fermented mixed extract powder prepared from the material was obtained (hereinafter referred to as 'F-CL' and the non-fermented mixed extract 'CL').

Reference Example 1. Preparation of Experimental Materials

3T3-L1 mouse-derived embryonic fibroblasts were purchased from American Type Culture Collection (ATCC, CL-173TM), Dulbeccos modified Eagles media (DMEM, SH30243.01), Bovine calf serum (BCS, SH30401.01) , Fetal bovine serum (FBS, SH30396.03) and penicillin and streptomycin (PS) were purchased from Hyclone (SV30010, Hyclone, USA). Insulin (090-03446), dimethyl sulfoxide (DMSO, D2650), Oil Red-O (O0625-25), dexamethasone (DEX, D4902-100MG) and isobutylmethyl Xanthine (isobutylmethylxanthine (IBMX, I5879-250MG) was purchased from Sigma-Aldrich company, St. Louis, Mo., USA and used.

Reference Example 2. Preparation of Laboratory Animals

Experimental animals were used 5 weeks old ICR mice (Orient Corp.) weighing 28-32 g in weight, and were fed in the animal breeding room of Daegu Haany University under constant conditions (temperature: 21 ± 2, contrast: 12 hour contrast cycle). Free intake of negative water was allowed, and sufficient water and food were provided until the start of the experiment.

Reference Example   3. 3 T3 - L1  Cell culture

3T3-L1 mouse embryo fibroblasts were cultured by the following experimental method (Lin. J. et al., Green Tea Polyphenol Epigallocatechin Gallate Inhibits Adipogenesis and Induces Apoptosis in 3T3-L1 Adipocytes, Obesity Research, 13 (6) , pp. 982-990, 2005).

First, the cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% bovine calf serum at 37 ° C in a 5% CO ₂ incubator (Day 0). After 8 days of incubation, preadipocytes were transferred to DMEM medium containing 167 nM insulin, 0.5 μΜ isobutylmethylxanthine and 1 μΜ dexamethasone containing 10% fetal bovine serum (FBS) for 2 days (Day 2). . 167 nM insulin for 2 days and then cultured in 10% FBS / DMEM for 4 days to produce mature adipocytes.

Reference Example   4. Administration of Fermented Gold Coin, Fermented Dermal Mixed Herbal Extract and Induction of Obesity

To induce obesity, experiments were conducted using the dietary compositions described in the literature (Lee JS et al., Supplementation of whole persimmon leaf improves lipid profiles and suppresses body weight gain in rat fed high-fat diet, Food and Chemical). Toxicology, 44 (11) , pp. 1875-1883, 2006).

First, in the experimental group except the normal group, a pellet-type solid feed (# 102038, AIN-76A Based High Fat / High Carb Purified) containing 1% cholesterol and 15% Lard was added to the diet based on the AIN-76 composition. Rodent Diet with Cholesterol & Cholic Acid, Dyet, USA) was fed for 6 weeks and 15 animals were assigned to each group. That is, divided into normal group (Normal), high fat control group (HF), high fat feed unfermented mixed extract (CL-5%), high fat feed fermented mixed extract (F-CL-5%), and the sample group in Example 1 The lyophilized mixed herbal extract powder obtained in was added to the high fat diet by 5% and fed with water every morning, and 20 g of feed were allocated daily.

Ingredient Nomal HF CL F-CL HCA Casein 200 200 199 199 200 DL - Methionine 3 3 3 3 3 Com starch 150 150 150 150 150 Sucrose 500 397.5 383 379 391.5 Cellulose 50 50 50 50 50 Corn oil 50 50 49.7 49.3 50 Mineral-mix 35 35 35 35 35 Vitamin-mix 10 10 10 10 10 Choline bitartrate 2 2 2 2 2 Lard - 100 99.8 99.2 100 Cholesterol - 2 2 2 2 Cholic acid - 0.5 0.5 0.5 0.5 CL: 5% (yield 32%) - - 16 - - F-CL: 5% (yield 42%) - - - 21 - Garcinia cambogia 1%
(HCA 0.6%) -
- - - 10 ¹⁾ total (%) 1000 1000 1000 1000 1000 Kcal / 100g diet 3850 4340 4274 4249 4316 Calorie from fat (%) 117 311 311 311 311 Calorie from carbohydrate (%) 675 505 505 505 505 Calorie from protein (%) 208 184 184 184 184

Reference Example   5. Statistical Processing

The statistical significance of the test results was analyzed using two-way ANOVA and LSD post-test of the SPSS program (Version 14.01) . In the case of 05 , it was recognized as significant, and the statistical significance (#) with the normal group and the high-fat diet administration group (HF) was shown in the figures and tables, respectively.

Experimental Example  1. Cytotoxicity MTT assay )

To determine the effect of fermentation, unfermentation, and mixed extracts on 3T3-L1 cells, MTT assay (methylthiazol tetrazolium bromide) was used (Lin J. et al., Green Tea Polyphenol Epigallocatechin Gallate Inhibits Adipogenesis and Induces Apoptosis in 3T3-L1 Adipocytes, Obesity Research 13 (6), pp. 982-990, 2005).

The MTT assay is a method of measuring the absorbance by dissolving blue formazan crystals produced by dehydrogenase in mitochondria of living cells with DMSO (dimethyl sulfoxide). The 3T3-L1 cell line cultured in Reference Example 1 was used, and cells in exponential growth phase were used in the experiment. In other words, 5 × 10 ⁴ cells / ml were mixed with 5% tryphan in the same volume for the same period without any treatment, and the number of living cells was measured. The viability was evaluated using the average value of three wells. Add 50 μl of MTT solution (2 mg / ml) per well and react for 2 hours at 37 ° C and 5% CO ₂ incubator. Remove the medium and add 100 μl of DMSO per well for 5 minutes. After dissolution, absorbance was measured at 540 nm of ELISA reader (Techan, Germany).

As a result, as shown in FIG. 1, fermented herbal medicine (F-CL) and unfermented herbal medicine (CL) were treated for 24 hours with different concentrations (0.1, 0.3, 1 mg / ml), and the MTT assay was tested. Compared with the sample concentration, the survival rate of the adipocytes was observed to be decreased, and cell viability was significantly inhibited than the fermented medicinal herb extract, especially at 0.1 mg / ml of fermented medicinal herbs (F-CL).

Experimental Example  2. Using cells oil red -O  dyeing

Oil Red-O staining experiments were performed to investigate the effects of fermentation, unfermentation, and mixed extracts on adipocyte differentiation and fat production in 3T3-L1 cells (Lin J. et al., Green Tea Polyphenol Epigallocatechin Gallate Inhibits Adipogenesis). and Induces Apoptosis in 3T3-L1 Adipocytes, Obesity Research 13 (6), pp.982-990, 2005).

Oil Red-O staining is a method of measuring the fat produced in the cells by staining with differentiated 3T3-L1 cells with Oil Red-O reagent. The medium of the differentiated cells was removed, the cells were washed with D-PBS (DPBS, SH30028.03), fixed with pH 7.2 Cacodylate buffer for 2 hours, and stained with Oil-red O Solution. After the cell staining was washed three times with 40% Isopropyl alcohol (2-Propanol, 67-63-0) and dried to observe the size of the fat in the cell under an optical microscope (AE 31, Motic, USA).

As shown in FIG. 2, the fermented and unfermented mixed extracts were treated with 100 μg / ml (B), 150 μg / ml (C), and 200 μg / ml (D), respectively, during differentiation of 3T3-L1 cells. As a result of treatment of the sample at concentration, it inhibited cell differentiation and lipogenesis in a concentration-dependent manner compared to the control (control). It was excellent in inhibiting fat accumulation.

Experimental Example  3. 3 T3 - L1 Cell Western

3T3-L1 Cell Western was performed to investigate the effects of fermented extracts on AMPK, ACC and PPAR-γ activity (Huang B. et, al, Cinnamaldehyde prevents adipocyte differentiation and adipogenesis via regulation of peroxisome proliferator-activated receptor- γ (PPARγ) and AMP-activated protein kinase (AMPK) pathways, J Agric Food Chem, Apr 27; 59 (8), pp. 3666-73, 2011).

After washing 3T3-L1 cells by concentration with D-PBS (DPBS, SH30028.03), 2 ml of 50 mM tris-HCl (pH7.5) buffer containing 1 mM EDTA was added to remove the cells, followed by Branson The model 185 sonifier was sonicated at 40 watts for 5 seconds. After centrifugation at 13,000 rpm for 4 minutes at 4 ° C, 20 μg of the same amount of protein is denatured and 8% or 10% SDS-PAGE is separated and transferred to PVDF membrane. The mebrane was blocked with 5% skin milk dissolved in TBS-T for 2 hours, and then treated with rabbit polyclonal anti-PPARγ, AMPK, and ACC antibody for 1 hour. After washing with TBS-T and treating with horseradish peroxidase secondary antibody for 1 hour and washing with TBS-T again, the immunorective bands were observed with ECL detection reagent.

Experimental results, as shown in FIG. The phosphorylation activity of AMPK was increased in the fermented extracts. In comparison, the unfermented mixed extract had no effect on the phosphorylation activity of AMPK. In addition, the phosphorylation activity of AMPK increased in proportion to the concentration of fermented mixed extract. ACC according to AMPK phosphorylation activity was also observed to be significantly activated in the fermentation mixed extract. In addition, PPAR-γ is a specific transcription factor in adipocytes that is expressed late in differentiation and plays an important role in the expression of several hormones, adipocytokine and adipogenic protein. As a result of confirming the inhibitory activity of PPAR-γ protein expression in the mixed fermented and unfermented extracts, the PPAR-γ was significantly decreased in the fermented extracts.

Experimental Example  4. High Fat Diet  Induction of Obesity-induced Rats Dietary efficiency , Weight measurement and tissue collection

Dietary intake was measured daily in the morning, daily food intake was calculated and body weight was measured once a week. The food efficiency ratio (FER) is the body weight gain during the experiment and the value divided by the dietary intake (FER = body weight gain / food intake).

As shown in Table 2 and FIG. 4, after 6 weeks, body weight was significantly increased in the high-fat diet group (HF) than in the normal group (NOR), and body weight was increased in the HCA, CL, and F-CL groups. Results in significantly suppressing Dietary intake was significantly decreased in HCA group, CL and F-CL group compared to normal group (NOR). In the change of body weight, the high fat diet was significantly decreased in the NOR, HCA and F-CL groups compared to the HF group. In FER, the HF, HCA, CL, and F-CL groups showed significantly higher results than the normal group.

On the other hand, the animals were fasted 15 hours before sacrifice and collected liver and site-specific visceral adipose tissue (epididymal, visceral adipose tissue) and brown adipose tissue (BAT) under CO ₂ anesthesia. . Tissue staining tissues were fixed in formalin solution, and the analytical body fat tissues were rapidly frozen with liquid nitrogen and stored at -80 ° C until use.

5 and 6 (ad), the liver weight, epididymal fat weight, peripheral fat weight, visceral weight, and brown fat (BAT) were significantly increased in the high fat diet group (HF) group compared to the normal group. In the HCA, CL and F-CL groups, hepatic weight, epididymal fat weight, peri-dial adipose tissue weight, and brown fat weight were decreased, especially in the F-CL group. This result shows that the effect of strong F-CL on body fat reduction, which is known to cause complications such as cardiovascular disease, rather than simple weight loss in obesity, has a greater significance as a treatment for obesity.

NOR HF HCA CL F-CL food intake
550 ± 0.14 ^* 5.04 ± 0.09 4.02 ± 0.07 ^{#, *} 4.28 ± 0.04 ^{#, *} 3.84 ± 0.16 ^{#, *} Weight gain (g / day)
0.17 ± 0.02 ^{#, *} 0.27 ± 0.02 0.09 ± 0.02 ^* 0.22 0.02 0.15 ± 0.01 ^* FER ^*
0.03 ± 0.00 ^* 0.05 ± 0.00 ^{#, *} 0.07 ± 0.00 ^# 0.05 ± 0.01 ^# 0.05 ± 0.00 ^#

Experimental Example  5. Blood collection and serum analysis of obese rats

Experimental animals of each group were fasted 15 hours before sacrifice, lightly anesthetized with CO ₂ , and blood was collected from the abdominal vena cava. The collected blood is placed in a test tube and allowed to stand at room temperature for 1 hour, followed by centrifugation at 4 ° C and 3,500 rpm for 15 minutes to separate serum. Total cholesterol (TC) in serum is determined by the enzyme method of C. Allain 1 (Allain 1). Analytical reagents (L-type GHO M, Wako, Japan) were used according to C. et al., Enzymatic determination of total serum cholesterol, Clin.Chem. 20 (4) , pp.470-475, 1974). The triglyceride content was determined by Van Handel et al. (Van Handel E. et al, Micromethod for the direct determination of serum triglycerides, J. Lab. Clin. Med. 50 (1) , pp.152-157, 1957). Test reagents (L-type TG M, Wako, Japan) were used. High-density lipoprotein (HDL-cholesterol) and low-density lipoprotein (LDL-cholesterol) concentrations were determined by Warnick's enzyme method (Warnick GR et al., Dextran sulfate-Mg ²⁺ precipitation procedure for quantitation of high density lipoprotein cholesterol, Clin. Chem. 20 (6) , pp. 1397-1388, 1982) using test reagents (Choesterol N HDL, Choesterol N LDL, SEKISUI, Japan). The content of Alanine aminotransferase (ALT) and serum Aspartate aminotransferase (AST) in transaminase related to liver function was measured by Giusti G. et al., A comparative study of some spectrophotometric and colorimetric procedures for the determination of serum glutamic-oxaloacetic and glutamic-pyruvic transaminase in hepatic diseases.Enzymol.Biol.Clin (Basel), 10 (1) , pp.17-38, 1969) J2, L-type AST J2, Wako, Japan) using an automated biochemistry analyzer (Hitachi 7080, Japan). Leptin concentration in serum was measured using Leptin ELISA kit (900-015, Assay Designs, USA), and adiponectin concentration in serum was determined using Adiponectin ELISA kit (44-ADPR-0434, ALPCO Diagnostics, USA). Analysis was performed with an ELISA reader (Tecan, Germany).

As shown in FIG. 7 (af), the ALT, AST and triglyceride concentrations and the low density lipoprotein (LDL) content in the serum were increased in the high fat diet group (HF) group compared to the normal group, but the administration of F-CL ALT, AST and triglyceride concentrations and low density lipoprotein (LDL) content in serum were decreased, and high density lipoprotein (HDL) content in serum was decreased in high fat diet group (HF) group compared to normal group. HDL content tended to increase.

Serum content of adiponectin secreted by adipocytes while acting as an insulin-sensitive hormone was increased in the normal group (NOR) than the high-fat diet group (HF) as shown in Figure 8-a, by F-CL administration The adiponectin content in the serum tended to be higher than that in the high fat diet group (HF).

As a peptide secreted by adipocytes, the serum content of leptin, known to be related to appetite control, was significantly increased in the high fat diet group (HF) compared to the normal group (NOR), as shown in FIG. 8-b. Leptin content in serum was significantly lower than that of high fat diet group (HF) by F-CL administration.

Experimental Example  6. In muscle AMPK  , ACC  And PPAR -γ activity

3T3-L1 Cell Western was performed to investigate the effects of fermented extracts on AMPK, ACC and PPAR-γ activity (Murase T. et, al, Nootkatone, a characteristic constituent of grapefruit, stimulates energy metabolism and prevents diet- induced obesity by activating AMPK, Am J Physiol Endocrinol Metab, 299 (2), pp. E266-75, 2010).

Tissue was used with RIPA buffer (150 mM NaCl, 0.5% Triton X-100, 50 mM Tris-HCl, pH 7.4, 25 mM NaF, 20 mM EGTA, 1 mM DTT, 1 mM Na ₃ VO ₄ , protease inhibitor cocktail) After homogenization, the supernatant was collected by centrifugation at 14,000 g for 10 minutes at 4 ° C., the protein content was quantified with BCA reagent, and 20 ㎍ was electrophoresed on 8% SDS-PAGE. The developed gel was transferred to PVDF membrane (Millipore) and blocked with 5% bovine serum albumin (BSA) -TBST (TBS, 0.1% Tween-20). 5% bovine serum albumin (BSA) -TBST in anti-ACC (1: 1000 dilution, Cell Signaling), P-ACC (1: 1000 dilution, Cell Signaling), anti-AMPK (1: 1000 dilution, Cell Signaling), P-AMPK (1: 1000 dilution, Cell Signaling), PPAR-γ (1: 1000 dilution, Santa cruz Biotechnology, USA) reacted with primary antibody overnight, washed 6 times with TBST solution for 5 minutes and then secondary antibody (goat anti -rabbit-HRP, 1: 5000 dilution, Assay designs) was added to 5% bovine serum albumin (BSA) -TBST for 1 hour and then washed 6 times with TBST solution for 5 minutes. After the reaction for 1 minute using ECL reagent (Amersham Pharmacia Biotech., Arlington Heights, IL, USA) and confirmed the protein attached to the Membrane using Image Quant software (Buckinghamshire, UK).

As a result, as shown in Figure 9, when the effects of fermented herbal extracts and unfermented herbal extracts on the AMPK, ACC phosphorylation in muscle related to obesity in the muscles confirmed by Western blot, the expression level of the band in P-AMPK Compared with the high fat diet control (HF), the activity of fermented herbal extracts (F-CL) increased. Like P-AMPK, P-ACC was activated in the F-CL group and significantly more active than the drug control group (HCA) when compared with the high-fat diet group (HF).

Hereinafter, the preparation examples of the composition including the extract of the present invention, but the present invention is not intended to limit it, but is intended to explain in detail only.

Formulation example  One. Sanje  Produce

Gold Silver Cod, Dermis Mixed Herbal Extract (F-CL) 20 mg

Lactose 100 mg

Talc 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

Formulation example  2. Preparation of tablets

Gold Silver Cod, Dermal Mixed Herbal Extract (F-CL) 10 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

Formulation example  3. Preparation of capsules

Gold Silver Cod, Dermis Mixed Herbal Extract (CL) 10 mg

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2 mg

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

Formulation example  4. Preparation of injections

Gold Silver Cod, Dermis Mixed Herbal Extract (CL) 10 mg

180 mg mannitol

Sterile sterilized water for injection 2974 mg

Na ₂ HPO ₄ , 12H2O 26 mg

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

Formulation example  5. Liquid  Produce

Gold Silver Cod, Dermis Mixed Herbal Extract (F-CL) 20 mg

10 g of isomerized sugar

5 g of mannitol

Purified water

Each component was added to purified water in accordance with the usual liquid preparation method and dissolved, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was adjusted to 100 ml with purified water, And sterilized to prepare a liquid preparation.

Formulation example  6. Manufacture of health food

Gold Silver Cod, Dermis Mixed Herbal Extract (F-CL) 1000 mg

Vitamin mixture proper amount

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

0.2 μg of vitamin B12

Vitamin C 10 mg

10 μg biotin

Nicotinic Acid 1.7 mg

50 μg folic acid

Calcium Pantothenate 0.5mg

Mineral mixture

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dibasic calcium phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

Formulation example  7. Manufacture of health drinks

Gold silver coin, dermis mixed herbal extract (CL) 1000mg

Citric acid 1000 mg

100 g of oligosaccharide

Plum concentrate 2 g

Taurine 1 g

Purified water was added to a total of 900 ml

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was heated at 85 DEG C for about 1 hour with stirring, and the solution thus prepared was filtered to obtain a sterilized 2-liter container, which was sealed and sterilized, ≪ / RTI >

Although the composition ratio is a composition that is relatively suitable for the preferred beverage in a preferred embodiment, the compounding ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and usage.

Claims (8)

A pharmaceutical composition for the prevention and treatment of obesity, containing fermented or unfermented sterling silver and dermis mixed herbal extracts as an active ingredient.
The method of claim 1,
The pharmaceutical composition, characterized in that the combination of the silver and silver: weight ratio of the dermis (w / w) is 1-5: 1 (w / w).
The method of claim 1,
The mixed herbal extract is a pharmaceutical composition, characterized in that the extract available in a solvent selected from water, lower alcohols having 1 to 4 carbon atoms or a mixed solvent thereof.
The method of claim 1,
The fermentation extract is a sterilization step of undergoing sterilization process for 10 minutes to 70 minutes at 80 to 150 ℃ the sample dried individual herbal medicines with a water content of less than 20%; Adding a fermentation strain to the sterilized sample in an amount of 1 to 30% of the sample weight to perform fermentation for 1 to 30 days at a temperature of 10 to 50 ° C. under a condition of 10 to 50% of moisture; A third step of drying the fermented product after completion of fermentation at a temperature of 30 to 100 ° C. for 12 to 72 hours; A fourth step of releasing the dried agglomerated particles to separate themselves and separating and selecting the fine particles; Pharmaceutical composition, characterized in that obtained in the process comprising a fifth step of performing the selected fermentation process for 5 minutes to 60 minutes with far-infrared herb at 100 to 300 ℃.
Health functional food for the prevention and improvement of obesity containing fermented or unfermented gold and silver dermis mixed herbal extract as an active ingredient.
The health functional food according to claim 5, wherein the health functional food is in the form of powder, granule, tablet, capsule, pill, tea bag, leach tea or health beverage.
A dietary supplement comprising as an active ingredient a mixed herbal extract of fermented or unfermented gold and silver and dermis exhibiting anti-obesity activity.
Sterilization step of sterilizing the sample of the gold and silver dermis individual herbal medicines dried to less than 20% moisture content at 10 to 70 minutes at 80 to 150 ℃; Adding a fermentation strain to the sterilized sample in an amount of 1 to 30% of the sample weight to perform fermentation for 1 to 30 days at a temperature of 10 to 50 ° C. under a condition of 10 to 50% of moisture; A third step of drying the fermented product after completion of fermentation at a temperature of 30 to 100 ° C. for 12 to 72 hours; A fourth step of releasing the dried agglomerated particles to separate themselves and separating and selecting the fine particles; Obtaining a fermentation sample by performing a fifth step of performing the process of subtracting the selected fermented product with a far-infrared herb for 5 to 60 minutes at 100 to 300 ° C; The fermentation sample or fermented dry herbal material obtained in the above step is washed with gold and silver dermis, and the gold silver: the weight mixing ratio (w / w) of the dermis is 1-5: 1 (w / w) and the 1 to 1 of the sample After performing hot water extraction, cold extraction or ultrasonic extraction for 20 hours (w / v) of water, methanol, ethanol or a mixed solvent thereof at 50 to 120 for 1 to 5 hours, the extract obtained is filtered through filter paper. A method for producing a mixed herbal extract of fermented or unfermented sterling silver and dermis of claim 1 by performing lyophilization, room temperature drying or hot air drying on the obtained filtrate.

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