WO2013022178A1 - Composition for the prevention and treatment of obesity containing an active ingredient in the form of a fermented or unfermented lonicera japonica and aurantii nobilis pericarpium mixed herbal-preparation extract, and a use therefor - Google Patents

Composition for the prevention and treatment of obesity containing an active ingredient in the form of a fermented or unfermented lonicera japonica and aurantii nobilis pericarpium mixed herbal-preparation extract, and a use therefor Download PDF

Info

Publication number
WO2013022178A1
WO2013022178A1 PCT/KR2012/004237 KR2012004237W WO2013022178A1 WO 2013022178 A1 WO2013022178 A1 WO 2013022178A1 KR 2012004237 W KR2012004237 W KR 2012004237W WO 2013022178 A1 WO2013022178 A1 WO 2013022178A1
Authority
WO
WIPO (PCT)
Prior art keywords
fermented
extract
obesity
fermentation
silver
Prior art date
Application number
PCT/KR2012/004237
Other languages
French (fr)
Korean (ko)
Inventor
김미려
이은실
Original Assignee
대구한의대학교 산학협력단
영농조합법인 이도
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 대구한의대학교 산학협력단, 영농조합법인 이도 filed Critical 대구한의대학교 산학협력단
Publication of WO2013022178A1 publication Critical patent/WO2013022178A1/en

Links

Images

Classifications

    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18Magnoliophyta (angiosperms)
    • A61K36/185Magnoliopsida (dicotyledons)
    • A61K36/35Caprifoliaceae (Honeysuckle family)
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18Magnoliophyta (angiosperms)
    • A61K36/185Magnoliopsida (dicotyledons)
    • A61K36/75Rutaceae (Rue family)
    • A61K36/752Citrus, e.g. lime, orange or lemon
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18Magnoliophyta (angiosperms)
    • A61K36/185Magnoliopsida (dicotyledons)
    • A61K36/35Caprifoliaceae (Honeysuckle family)
    • A61K36/355Lonicera (honeysuckle)
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00Drugs for disorders of the metabolism
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00Drugs for disorders of the metabolism
    • A61P3/04Anorexiants; Antiobesity agents

Definitions

  • the present invention relates to a composition for the prevention and treatment of obesity and its use containing fermented or unfermented sterling silver and dermis mixed herbal extracts as an active ingredient.
  • Cinnamaldehyde prevents adipocyte differentiation and adipogenesis via regulation of peroxisome proliferator-activated receptor- ⁇ (PPAR ⁇ ) and AMP-activated protein kinase (AMPK) pathways, J Agric Food Chem, Apr 27; 59; (8) , pp. 3666-73, 2011
  • PPAR ⁇ peroxisome proliferator-activated receptor- ⁇
  • AMPK AMP-activated protein kinase
  • 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.
  • Metabolic disease is estimated to increase by about 50% to about 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). All inde trend worldwide increase in overweight or obese people that has been a significant increase in the incidence of obesity in our country 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. Cellutrient intake and obesity. J Food Sci Nutr. 5 (1) , pp.58-64, 2000).
  • 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.
  • 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.
  • Fermented herbal medicine refers to herbal medicines fermented using purely isolated microorganisms such as microorganisms in the air or lactic acid bacteria after steaming or boiling traditional herbal medicines so that microorganisms can use them well through traditional fermentation methods. 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.
  • various manufacturing methods and fermented herbal medicines are being developed, and fermented red ginseng products and fermented herbal cosmetics can be considered as representative products.
  • the mixed herbal extract obtained by fermenting sterling silver and dermis can be used as a composition for preventing and treating obesity.
  • 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.
  • 3T3-L1 is a fat cell
  • adiponectin adiponectin
  • 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%
  • 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 °C, preferably 150 to 200 °C Obtainable.
  • 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 °C 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.
  • 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 present invention also provides a method for treating obese patients, comprising administering a mixed herbal extract of fermented or unfermented sterling silver and dermis to an obese patient.
  • the present invention also provides the use of a mixed herbal extract of fermented or unfermented sterling silver and dermis for the manufacture of a medicament for the treatment of obesity.
  • 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.
  • compositions comprising the extract of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.
  • compositions 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.
  • 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, 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 sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories.
  • 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, 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. 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.
  • 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.
  • 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.
  • 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 It means ingestion for the purpose of obtaining useful effects on health use such as nutrient control or physiological action on structure and function.
  • the extract comprises 0.01 to 95% by weight, preferably 1 to 80% by weight relative to the total weight of the composition.
  • a pharmaceutical dosage form such as powders, granules, tablets, capsules, pills, suspensions, emulsions, syrups, or health functional foods in the form of tea bags, leach teas, and health drinks. 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 is not particularly limited to other ingredients except for containing the extract as an essential ingredient in the indicated proportions, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks.
  • 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.
  • natural flavoring agents such as, tauumatin, stevia extract (e.g., Rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used.
  • the proportion of the natural carbohydrate is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.
  • 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 from the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.
  • the extract of the present invention may be added to food or beverage for the purpose of preventing obesity.
  • 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.
  • 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,
  • FIG. 2 is a diagram showing the effect of F-CL on Oil Red-O staining of 3T3-L1, a cell line of adipocytes,
  • FIG. 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
  • FIG. 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 (a-d) 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 (a-c) is a diagram showing the effect of F-CL on the blood lipid concentration of obese mice due to high-fat diet administration
  • Figure 8 (d-f) is a diagram showing the effect of F-CL on the blood lipid concentration of obese mice due to high-fat diet administration
  • FIG. 9 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,
  • FIG. 10 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.
  • the above ingredients are mixed and filled in an airtight cloth to prepare a powder.
  • tablets are prepared by tableting according to a conventional method for preparing tablets.
  • the above ingredients are mixed and filled into gelatin capsules to prepare capsules.
  • the amount of the above ingredient is prepared per ampoule (2 ml).
  • each component is added to the purified water to dissolve it, the lemon flavor is added appropriately, the above components are mixed, purified water is added, the whole is adjusted to 100 ml by the addition of purified water, and then filled in a brown bottle.
  • the solution is prepared by sterilization.
  • Vitamin B6 0.5 mg
  • composition ratio of the above-mentioned vitamin and mineral mixtures is mixed with a component suitable for a health food in a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method.
  • the granules may be prepared and used for preparing a health food composition according to a conventional method.
  • composition ratio is a composition that is relatively suitable for the preferred beverage in a preferred embodiment
  • compounding ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and usage.
  • Fermented gold silver, dermis, unfermented gold silver and dermis fermented by the above process was measured 5 kg in a 3: 2 weight ratio, and then distilled water of 10 times and then heated to 95 °C and extracted for 12 hours. After extraction, the precipitate was filtered using a filter paper (Advantec, 71101903), and the filtrate was concentrated under reduced pressure in a rotary vacuum distillation machine (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 fermented mixed extract 'CL').
  • 3T3-L1 mouse-derived embryonic fibroblasts were purchased from the 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.
  • mice 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.
  • 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).
  • DMEM Dulbecco's modified Eagle's medium
  • DMEM bovine calf serum
  • FBS fetal bovine serum
  • 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.
  • MTT assay methylthiazol tetrazolium bromide
  • 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).
  • DMSO dimethyl sulfoxide
  • the 3T3-L1 cell line cultured in Reference Example 1 was used, and the cells in exponential growth phase were used for the experiment. In other words, 5 ⁇ 10 4 cells / ml was mixed well with the same amount of 5% tryphan without any treatment for a certain period of time, and the number of living cells was measured.
  • the viability test was evaluated using the average value of all three wells. Add 50 ⁇ l 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 ⁇ l of DMSO per well for 5 minutes. After dissolution, absorbance was measured at 540 nm of ELISA reader (Techan, Germany).
  • 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.
  • concentrations 0.1, 0.3, 1 mg / ml
  • MTT assay was tested.
  • 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).
  • Oil Red-O staining is a method of measuring the amount of intracellular fat produced by staining 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).
  • the fermented and unfermented mixed extracts were treated with 100 ⁇ g / ml (B), 150 ⁇ g / ml (C), and 200 ⁇ g / ml (D), respectively, during differentiation of 3T3-L1 cells.
  • B 100 ⁇ g / ml
  • C 150 ⁇ g / ml
  • D 200 ⁇ g / ml
  • 3T3-L1 Cell Western was performed to investigate the effects of fermented extracts on AMPK, ACC and PPAR- ⁇ activity (Huang B. et, al, Cinnamaldehyde prevents adipocyte differentiation and adipogenesis via regulation of peroxisome proliferator-activated receptor- ⁇ (PPAR ⁇ ) and AMP-activated protein kinase (AMPK) pathways, J Agric Food Chem, Apr 27; 59 (8), pp. 3666-73, 2011).
  • PPAR ⁇ peroxisome proliferator-activated receptor- ⁇
  • AMPK AMP-activated protein kinase
  • FER body weight gain / food intake
  • liver and site-specific visceral adipose tissue epididymal, visceral adipose tissue
  • brown adipose tissue BAT
  • the liver weight, epididymal fat weight, peripheral fat weight, visceral weight, and brown fat were significantly increased in the high fat diet group (HF) group compared to the normal group.
  • HF high fat diet group
  • 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.
  • 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.
  • ALT, AST and triglyceride concentrations and low density lipoprotein (LDL) content in serum were increased in the high fat diet group (HF) group compared to the normal group.
  • ALT, AST and triglyceride concentrations and low density lipoprotein (LDL) content in serum were decreased by administration of F-CL, and high density lipoprotein (HDL) content in serum was decreased in high fat diet group (HF) group compared to normal group.
  • F-CL administration tended to increase HDL content.
  • the serum content of adiponectin secreted from adipocytes while acting as an insulin-sensitive hormone was increased in the normal group (NOR) than the high-fat diet group (HF), by F-CL administration.
  • the adiponectin content in the serum tended to be higher than that in the high fat diet group (HF).
  • HF high fat diet administered group
  • NOR normal group
  • 3T3-L1 Cell Western was performed to investigate the effects of fermented extracts on AMPK, ACC and PPAR- ⁇ 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 ⁇ g 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).
  • BSA bovine serum albumin
  • 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.
  • 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Engineering & Computer Science (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • Alternative & Traditional Medicine (AREA)
  • Epidemiology (AREA)
  • Mycology (AREA)
  • Microbiology (AREA)
  • Medical Informatics (AREA)
  • Botany (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Hematology (AREA)
  • Obesity (AREA)
  • Diabetes (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Child & Adolescent Psychology (AREA)
  • Medicines Containing Plant Substances (AREA)
  • Coloring Foods And Improving Nutritive Qualities (AREA)

Abstract

The present invention relates to a composition for the prevention and treatment of obesity containing an active ingredient in the form of a fermented or unfermented Lonicera japonica and Aurantii Nobilis Pericarpium mixed herbal-preparation extract. The herbal-preparation extract of the present invention can advantageously be used in pharmaceuticals or in health and functional foods for the prevention and treatment of obesity, since the extract of the present invention is effective in suppressing adipose differentiation of 3T3-L1 cells which are mouse-derived adipocytes, and, in white rats in which obesity was induced by using a high-fat diet, the extract of the present invention is effective in reducing body weight and reducing body fat and in reducing the blood lipid concentration.

Description

발효 또는 미발효된 금은화 및 진피 혼합생약 추출물을 유효성분으로 함유하는 비만증의 예방 및 치료용 조성물 및 이의 용도Composition and its use for the prevention and treatment of obesity containing fermented or unfermented sterling silver and dermis mixed herbal extract as an active ingredient
본 발명은 발효 또는 미발효된 금은화 및 진피 혼합생약 추출물을 유효성분으로 함유하는 비만증의 예방 및 치료용 조성물 및 이의 용도에 관한 것이다.The present invention relates to a composition for the prevention and treatment of obesity and its use containing fermented or unfermented sterling silver and dermis mixed herbal extracts as an active ingredient.
[문헌 1] Van der Ploeg LH. Related Obesity: an epidemic in need of therapeutics. Curr Opin Chem Biol. 4(4), pp.452-460, 2004 Document 1 Van der Ploeg LH. Related Obesity: an epidemic in need of therapeutics. Curr Opin Chem Biol. 4 (4) , pp.452-460, 2004
[문헌 2] Kim DM, et al., Prevalence of obesity in Korea. Obes Rev. 6, pp.117-121, 2005[2] Kim DM, et al., Prevalence of obesity in Korea. Obes Rev. 6 , pp. 117-121, 2005
[문헌 3] Daily JW, Cha YS. Macronutrient intake and obesity. J Food Sci Nutr. 5(1), pp.58-64, 2000[Reference 3] Daily JW, Cha YS. Macronutrient intake and obesity. J Food Sci Nutr. 5 (1) , pp. 58-64, 2000
[문헌 4] 장개빈. 장씨유경. 서울, 성보사. p.586, 1982[Document 4] Jang Ga-bin. Mr. Zhang. Seongbosa, Seoul. p.586, 1982
[문헌 5] 김은선 외 2, 비만치료제, 심층정보분석 보고서, 한국과학기술정보연구원, pp.1-111, 2002[Document 5] Eun-Seon Kim et al. 2, Obesity Therapeutics, In-depth Information Analysis Report, KIST
[문헌 6] 전국한의과대학 공동교재편찬위원회, 본초학, 영림사, pp.242-244, 2006[Reference 6] National Institute of Oriental Medicine Joint Textbook Compilation Committee, Herbology, Younglim History, pp.242-244, 2006
[문헌 7] 전국한의과대학 공동교재편찬위원회, 본초학, 영림사, pp.466-469, 2006[Reference 7] National Institute of Oriental Medicine, Joint Textbook Editing Committee, Herbology, Younglimsa, pp.466-469, 2006
[문헌 8] 박정연, 산사은국차가 과체중여대생의 체중 혈청 지질 및 산화적 스트레스에 미치는 영향, 석사학위논문, 명지 대학교, 2009[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
[문헌 9] 최종환 외 7, 진피 복합제 복용과 운동이 비만 여성의 체격, 체지방 및 혈중지질에 미치는 영향, 생약학회지, 33(1), pp. 57-63, 2002[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
[문헌 10] 박치덕 , Aureobasidium pullulans를 이용한 진피 flavonoid의 생물전환 및 항비만 효능에 관한 연구, 박사학위논문, 계명대학교, 2011[Document 10] Park, Chi-Duk, A Study on the Bioconversion and Anti-obesity Effects of Dermal Flavonoid Using Aureobasidium pullulans , Ph.D. Thesis, Keimyung University, 2011
[문헌 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)[Document 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 )
[문헌 12] Wolfe K. et al., Antioxidant activity of apple peels. J. Agric. Food. Chem. 51, pp.609-614, 200312. Wolfe K. et al., Antioxidant activity of apple peels. J. Agric. Food. Chem. 51 , pp. 609-614, 2003
[문헌 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[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
[문헌 14] Lee J. S. 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, 2006Lee 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
[문헌 15] 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, 2011Huang 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
[문헌 16] Allain C. et al., Enzymatic determination of total serum cholesterol, Clin. Chem. 20(4), pp.470-475, 1974[16] Allain C. et al., Enzymatic determination of total serum cholesterol, Clin. Chem. 20 (4) , pp. 470-475, 1974
[문헌 17] Van Handel E. et al, Micromethod for the direct determination of serum triglycerides, J. Lab. Clin. Med. 50(1), pp.152-157, 195717. Van Handel E. et al, Micromethod for the direct determination of serum triglycerides, J. Lab. Clin. Med. 50 (1), pp.152-157, 1957
[문헌 18] Warnick G. R. et al., Dextran sulfate-Mg2+ precipitation procedure for quantitationof high density lipoprotein cholesterol, Clin. Chem. 20(6), pp.1397-1388, 198218. 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
[문헌 19] 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, 1969Giusti 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
[문헌 20] 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, 1998DiGirolamo 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
비만은 에너지의 섭취와 소비의 불균형에 의해서 야기되는 현상으로 체내에 지방이 과잉 축적되어 있는 상태를 의미한다. 다양한 원인에 의해 야기되기 때문에 명확한 원인은 아직 밝혀지지 않았지만 일반적으로 지나친 열량 섭취, 내분비 장애, 운동부족, 유전적 요인 등에 기인하는 것으로 인식되고 있다. 그리고 생활 습관과 밀접한 관계가 있기 때문에 일반적으로 생활습관병이라고도 한다. 국내에선 2000년 이후 심장병, 당뇨병 등과 같은 만성질환에 걸리기 쉬운 대사증후군을 갖고 있는 사람이 최근 5년 사이 65% 급증하고 있다. 이로 인하여 환자 본인의 고통 및 삶의 질 저하는 물론이고 사회적으로도 과도한 의료비 지출과 생산성 저하 등 다양한 문제점 발생하고 있다. 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.
세계보건기구(WHO)의 통계에 따르면, 2005년 현재 전 세계 인구 중에서 10억 명 정도가 비만인 것으로 추정하고 있으며, 전체 사망원인의 30% 이상이 비만을 비롯한 대사성질환과 관련된 것 또한, 향후 10년 이내에 대사성질환은 15억명 정도로 약 50% 증가될 것으로 추정하고 있다. 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. Metabolic disease is estimated to increase by about 50% to about 1.5 billion people.
비만은 건강을 해칠 정도로 체내 지방이 과도하게 축적된 상태를 말하며, 각 개인의 표준체중보다 20% 이상의 증가가 있을 때 비만이라고 정의할 수 있다. 구체적으로 지방의 무게가 체중에서 차지하는 체지방 비율이 남자의 경우는 25% 이상, 여자의 경우, 30% 이상일 때를 말한다(Van der Ploeg LH. Related Obesity: an epidemic in need of therapeutics. Curr Opin Chem Biol. 4(4), pp.452-460, 2004). 전 세계적으로 과체중 혹은 비만에 해당하는 사람들이 증가하는 추세인데 최근 우리나라에서도 비만의 발생률이 크게 증가하고 있다(Kim DM, et al., Prevalence of obesity in Korea. Obes Rev. 6, pp.117-121, 2005). 비만은 에너지 섭취와 소비의 불균형으로 인해 여분의 에너지가 지방으로 전환되므로 당뇨병, 고혈압, 심혈관계질환, 관절질환, 폐질환 및 일부 암 등의 다양한 퇴행성 질환들의 유병율과 밀접한 관계가 있고, 비만으로 야기되는 재정적 부담과 인명적 손실은 막대하다(Daily JW, Cha YS. Macronutrient intake and obesity. J Food Sci Nutr. 5(1), pp.58-64, 2000). 특히 동양인의 경우, 서양인에 비해 체질량 지수가 적어도 복부 비만이 심하여 고혈압, 당뇨병, 고지혈증과 같은 동맥관련 질환으로 인한 합병증의 감수성이 높기 때문에 비만관리가 더욱 중요시 된다. 한의학에서는 비만의 원인을 膏粱珍味와 기허, 습체 등으로 보고 있으며, 치료법으로는 이수화습, 化痰, 熱通腑, 活血祛瘀의 약물요법을 쓰고 있다(張介賓. 장씨유경. 서울, 성보사. p.586, 1982). 비만치료법으로는 식욕억제제, 지방흡수억제제, 에너지대사촉진제, 호르몬제제 등의 약물요법과 위절제술, 지방흡입술 등의 외과적 수술법이 사용되고 있으나, 치료효과의 지속적 유지 여부 및 약물 중단 시 체중이 증가하는 현상 등이 문제되고 있다(Van der Ploeg LH. Related Obesity: an epidemic in need of therapeutics. Curr Opin Chem Biol. 4(4), pp.452-460, 2004; 김은선 외 2, 비만치료제, 심층정보분석 보고서, 한국과학기술정보연구원, pp.1-111, 2002). 현재 의료계에서는 식욕억제제인 시부트라민과 지방흡수억제제인 올리스태트 등을 장기간 사용 가능한 비만약물로 인정하고 있지만 아직도 부작용에 대한 의문이 제기되고 있으므로 안전하게 체중을 감량하기 위해서 식이요법, 운동요법 및 행동요법을 포함하는 다각적 접근방법이 권장되고 있다. 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). All inde trend worldwide increase in overweight or obese people that has been a significant increase in the incidence of obesity in our country 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 the causes of obesity, deprivation, and humectants.Therapeutics are used to treat hydration, hydration, 痰 通腑, and activism. (張介賓. .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.
금은화(Lonicerae Flos)는 인동과(Caprifoliaceae)에 속하는 다년생 반상록의 덩굴성식물이며, 여름철 꽃이 피기 전에 채취하여 그늘에 말려 사용한다. 성분으로는 tannin, inositol, sterol, chlorogenic acid, isochlogenic acid등이 보고되어 있으며, flavonoid 성분으로는 luteolin, apigenin, luteolin-7-O-rhamnoglucoside. lonicerin, inositol, 정유, quercetin등을 함유하고 있다. 맛이 달고 약성이 차가워서 청열(淸熱)하고 해독하는 효능이 있어 열독창옹(熱毒瘡擁)을 치료하는 중요한 약이다(전국한의과대학 공동교재편찬위원회, 본초학, 영림사, pp.242-244, 2006). 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).
진피(Citri Reticullatae Pericarpium)는 운향과에 속하는 상록 소교목인 귤나무 및 같은 속 근연식물의 성숙한 과실의 열매껍질을 건저혼 것으로, 11월에 채취하여 햇볕에 말린다. 성분으로 flavonoid로서 hesperidin, naringin poncirin, nobilein, neoesprin, nobileting, neohesprindin, tanderetin 등을 함유한다. 맛은 맵고 쓰며, 약성이 따뜻하고, 방향성이 있으며, 주로 비(脾), 폐경(肺經)에 들어가서 효능을 발휘한다. 매운맛은 발산(發散)하고 행체(行體)하며, 맛이 쓰고 약성이 따뜻하여 조습거한(燥濕去寒)한다. 그런데, 체(滯)한 기(氣)를 행하게 하면 비위장(脾胃腸)이 스스로 건전하게 활동하게 되고, 비위(脾胃)의 한습(寒濕)이 제거되면 담연(淡延)이 저절로 없어지므로, 본품은 이기, 건비(建脾), 조습(操濕), 화담(化痰)하는 중요한 약이된다(전국한의과대학 공동교재편찬위원회, 본초학, 영림사, pp.466-469, 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 to win, dry, control, talk, etc. (National Medical University Compilation Committee, Herbology, Younglim History, pp.466-469, 2006).
최근 한약학계에서는 발효한약에 대한 관심이 증폭되고 있다. 발효한약은 전통발효공법을 통해 전통 한약재를 미생물이 잘 이용할 수 있게 찌거나 삶은 다음, 공기 중의 미생물 또는 혹은 유산균과 같은 순수 분리 미생물을 이용하여 발효한 한약재를 말한다. 이는 한약재 약효성분의 체내흡수율과 생체 이용률을 무도 극대화시킨 일종의 가공방법으로 약리적 기능성뿐만 아니라 한약의 제형개량과 포제방법을 향상시킬 수 있고 이를 통해 한약의 새로운 수요를 창출하고 고부가치의 새로운 한약제품을 개발할 수 있다는데 그 의의를 두고 있다. 현재 다양한 제법과 형태의 발효 한약이 개발되고 있으며 특히 발효 홍삼제품과 발효 한방 화장품 등을 그 대표적 제품으로 꼽을 수 있다.Recently, the interest in fermented herbal medicine is increasing in the herbal medicine world. Fermented herbal medicine refers to herbal medicines fermented using purely isolated microorganisms such as microorganisms in the air or lactic acid bacteria after steaming or boiling traditional herbal medicines so that microorganisms can use them well through traditional fermentation methods. 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, various manufacturing methods and fermented herbal medicines are being developed, and fermented red ginseng products and fermented herbal cosmetics can be considered as representative products.
하지만, 상기 어느 문헌에서도 금은화 및 진피를 발효시킨 혼합 생약 추출물이 비만의 예방 및 치료용 조성물로서 사용 가능하다고 교시되거나 개시된 바 없다. 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.
이에 본 발명자들은 비만에 효과적인 치료제를 개발하기 위한 연구를 통해 금은화 및 진피 발효 추출물이 지방세포인 3T3-L1의 분화를 억제하며, 고지방 식이를 이용한 비만 유도 동물에서 체중 및 체지방 감소, 혈중 지질농도의 감소뿐만 아니라 렙틴(leptin), 아디포넥틴(adiponectin)의 혈중 농도 감소를 가져왔으며, 부고환 지방세포의 직경을 감소시키는 것을 확인함으로써 본 발명을 완성하였다.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.
상기 목적을 달성하기 위하여, 본 발명은 발효 또는 미발효된 금은화 및 진피 혼합생약 추출물을 유효성분으로 함유하는 비만증의 예방 및 치료용 약학조성물을 제공한다.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.
또한, 본 발명은 발효 또는 미발효된 금은화 및 진피 혼합생약 추출물을 유효성분으로 함유하는 비만증의 예방 및 개선용 건강기능식품을 제공한다. 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.
본원에서 정의되는 혼합 생약 추출물은 물, 탄소수 1 내지 4의 저급알코올 또는 이들의 혼합용매로부터 선택된 용매에 가용한 추출물, 바람직하게는 물에 가용한 추출물을 포함한다.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.
본원에서 정의되는 혼합 생약의 바람직한 중량 배합비로는 금은화 : 진피의 중량 혼합비(w/w)가 1-5 : 1, 보다 바람직하게는 1-3 : 1(w/w)로 배합된 배합물을 포함하는 것임을 특징으로 한다. 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.
본원에서 정의되는 발효 추출물은 개개 생약을 수분 함수율이 20% 미만, 바람직하게는 10% 미만으로 건조시킨 시료를 80 내지 150℃에서 10분 내지 70분간, 바람직하게는 20 내지 30분간 멸균과정을 거치는 멸균단계; 멸균된 시료를 아스페르질러스 오리자에(Aspergillus oryzae), 아스파질러스 나이거(Aspergillus niger), 락토바실러스 아시도플러스(Lactobacillus acidophilus) 락토바실러스 카제이(Lactobacillus casei), 기타 식품가공용으로 가능한 발효균주, 바람직하게는 아스페르질러스 오리자에(Aspergillus oryzae)를 시료 중량의 1 내지 30%, 바람직하게는 1 내지 20% 양으로 첨가하여 수분 10 내지 50%, 바람직하게는 20 내지 40%의 조건에서 10 내지 50℃, 바람직하게는 20 내지 40℃ 온도에서 1일 내지 30일, 바람직하게는 5일 내지 20일, 보다 바람직하게는 8일 내지 12일간 발효를 수행하는 제 2단계; 상기 발효가 완료된 발효물을 30 내지 100℃, 바람직하게는 40 내지 60℃ 온도에서 12시간 내지 72시간, 바람직하게는 24시간 내지 52시간 동안 건조시키는 제 3단계; 상기 건조된 덩어리진 입자를 비벼 자체 분리되도록 풀어주고 미세입자를 분리 및 선별하는 제 4단계; 상기 선별된 발효물을 100 내지 300℃, 바람직하게는 150 내지 200℃에서 원적외선 초제로 5분 내지 60분간, 바람직하게는 10분 내지 30분간 덖음과정을 수행하는 제 5단계 공정을 포함하는 공정으로 수득가능하다.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.
이하, 본 발명을 상세히 설명한다. Hereinafter, the present invention will be described in detail.
본 발명의 혼합 생약 추출물은 하기와 같이 수득될 수 있다. The mixed herbal extract of the present invention can be obtained as follows.
본 발명의 발효 또는 미발효 혼합 생약 추출물은 먼저, 금은화 또는 진피 개개 생약을 수분 함수율이 20% 미만, 바람직하게는 10% 미만으로 건조시킨 시료를 80 내지 150℃에서 10분 내지 70분간, 바람직하게는 20 내지 30분간 멸균과정을 거치는 멸균단계; 멸균된 시료를 아스페르질러스 오리자에(Aspergillus oryzae), 아스파질러스 나이거(Aspergillus niger), 락토바실러스 아시도플러스(Lactobacillus acidophilus) 락토바실러스 카제이(Lactobacillus casei), 기타 식품가공용으로 가능한 발효균주를 시료 중량의 1 내지 30%, 바람직하게는 1 내지 20% 양으로 첨가하여 수분 10 내지 50%, 바람직하게는 20 내지 40%의 조건에서 10 내지 50℃, 바람직하게는 20 내지 40℃ 온도에서 1일 내지 30일, 바람직하게는 5일 내지 20일, 보다 바람직하게는 8일 내지 12일간 발효를 수행하는 제 2단계; 상기 발효가 완료된 발효물을 30 내지 100℃, 바람직하게는 40 내지 60℃ 온도에서 12시간 내지 72시간, 바람직하게는 24시간 내지 52시간 동안 건조시키는 제 3단계; 상기 건조된 덩어리진 입자를 비벼 자체 분리되도록 풀어주고 미세입자를 분리 및 선별하는 제 4단계; 상기 선별된 발효물을 100 내지 300℃, 바람직하게는 150 내지 200℃에서 원적외선 초제로 5분 내지 60분간, 바람직하게는 10분 내지 30분간 덖음과정을 수행하는 제 5단계 공정을 수행하여 본 발명의 발효 시료를 수득가능하다.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.
상기 단계에서 얻은 발효 시료 또는 미발효된 건조생약재료인 금은화 및 진피을 세척하여, 금은화 : 진피의 중량 혼합비(w/w)가 1-5 : 1, 보다 바람직하게는 1-3 : 1 (w/w)로 배합하고 상기 시료의 1 내지 20배 (w/v) 중량, 바람직하게는 1 내지 10배 (w/v) 중량의 물, 메탄올, 에탄올 또는 이들의 혼합용매, 바람직하게는 물로 50 내지 120℃, 바람직하게는 약 80-100℃에서 1시간 내지 5시간, 바람직하게는 2시간 내지 4시간 동안 열수 추출법, 냉침 추출법 또는 초음파 추출법, 바람직하게는 열수 추출법을 수행한 후에 얻어진 추출액을 여과지로 여과한 후에 얻어진 여과물을 동결건조, 상온건조 또는 열풍건조, 바람직하게는 동결건조를 수행하여 본 발명의 금은화, 진피로 구성된 발효 또는 미발효 혼합 생약 추출물을 수득할 수 있다. 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.
본 발명은 상기의 제조방법 및 상기 제조공정으로 얻어진 발효 또는 미발효된 금은화 및 진피 생약 추출물을 유효성분으로 함유하는 비만증 치료 및 예방을 위한 약학 조성물을 제공한다. 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.
또한 본 발명은 발효 또는 미발효된 금은화 및 진피의 혼합 생약 추출물을 비만증 환자에게 투여함을 포함하는, 비만증 환자를 치료하기 위한 치료방법을 제공한다. The present invention also provides a method for treating obese patients, comprising administering a mixed herbal extract of fermented or unfermented sterling silver and dermis to an obese patient.
또한 본 발명은 비만증 치료용 약제를 제조하기 위한 발효 또는 미발효된 금은화 및 진피의 혼합 생약 추출물의 용도를 제공한다.The present invention also provides the use of a mixed herbal extract of fermented or unfermented sterling silver and dermis for the manufacture of a medicament for the treatment of obesity.
상기에서 수득된 금은화, 진피 생약 추출물은 지방세포의 지방 분화를 감소시켜 주며, 고지방 식이를 이용해 비만을 유도한 흰쥐에서 체중감소 및 체지방 감소, 혈중지질농도 감소효과를 나타낸다.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.
본 발명의 비만증 치료용 약학조성물은, 조성물 총 중량에 대하여 상기 추출물을 0.01 내지 80 중량%, 바람직하게는 10 내지 50 중량%로 포함한다. 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.
본 발명의 추출물을 포함하는 약학조성물은 약학적 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제 및 희석제를 더 포함할 수 있다.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.
본 발명의 추출물의 약학적 투여 형태는 이들의 약학적 허용가능한 염의 형태로도 사용될 수 있고, 또한 단독으로 또는 타 약학적 활성 화합물과 결합뿐만 아니라 적당한 집합으로 사용될 수 있다.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.
본 발명에 따른 추출물을 포함하는 약학조성물은, 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있다. 추출물을 포함하는 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로즈, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무,알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로즈, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수있다. 제제화 할 경우에는 보통 사용하는 충진제, 중량제, 결합제, 습윤제, 봉해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 추출물 및 분획물에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트(calcium carbonate), 수크로스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용된다. 경구를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제, 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세롤젤라틴 등이 사용될 수 있다.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 the 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 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, glycerol gelatin and the like can be used.
본 발명의 추출물을 포함하는 약학조성물의 바람직한 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 기간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다. 그러나 바람직한 효과를 위해서, 본 발명의 추출물을 포함하는 약학조성물은 1일 0.0001 내지 100 mg/kg으로, 바람직하게는 0.001 내지 100 mg/kg으로 투여하는 것이 좋다. 투여는 하루에 한번 투여할 수도 있고, 수회 나누어 투여할 수도 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다. 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. 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.
본 발명의 발효 또는 미발효된 금은화 및 진피 추출물은 쥐, 생쥐, 가축, 인간 등의 포유동물에 다양한 경로로 투여될 수 있다. 투여의 모든 방식은 예상될 수 있는데, 예를 들면, 경구, 직장 또는 정맥, 근육, 피하, 자궁내 경막 또는 뇌혈관내(Intracerebroventricular) 주사에 의해 투여될 수 있다. 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.
또한, 본 발명은 비만에 대하여 탁월한 억제효과를 갖는 발효 또는 미발효된 금은화 및 진피 혼합 생약 추출물을 유효성분으로 함유하는 비만증의 예방 및 개선을 위한 건강기능식품을 제공한다.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.
본원에서 정의되는 "건강기능식품"은 건강기능식품에 관한 법률 제6727호에 따른 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 제조 및 가공한 식품을 의미하며, "기능성"이라 함은 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건 용도에 유용한 효과를 얻을 목적으로 섭취하는 것을 의미한다.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 the Health Functional Food Act No. 6767, 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.
본 발명의 비만증의 예방 및 개선을 위한 건강기능식품은, 조성물 총 중량에 대하여 상기 추출물을 0.01 내지 95%, 바람직하게는 1 내지 80% 중량백분율로 포함한다.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.
또한, 비만증의 예방 및 개선을 위한 목적으로 산제, 과립제, 정제, 캡슐제, 환제, 현탁액, 에멀젼, 시럽 등의 약학 투여형태 또는 티백제, 침출차, 건강 음료 등의 형태인 건강기능식품으로 제조 및 가공이 가능하다.In addition, for the purpose of preventing and improving obesity, it is prepared as a pharmaceutical dosage form such as powders, granules, tablets, capsules, pills, suspensions, emulsions, syrups, or health functional foods in the form of tea bags, leach teas, and health drinks. Processing is possible.
또한, 본 발명은 비만증 억제활성을 나타내는 발효 또는 비발효된 금은화 및 진피의 혼합 생약 추출물을 주성분으로 함유하는 건강보조식품을 제공한다.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.
본 발명의 건강 기능성 음료 조성물은 지시된 비율로 필수 성분으로서 상기 추출물을 함유하는 외에는 다른 성분에는 특별한 제한이 없으며 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물의 예는 모노사카라이드, 예를 들어, 포도당, 과당 등; 디사카라이드, 예를 들어 말토스, 슈크로스 등; 및 폴리사카라이드, 예를 들어 덱스트린, 시클로덱스트린 등과 같은 통상적인 당, 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 상술한 것 이외의 향미제로서 천연 향미제(타우마틴, 스테비아 추출물(예를 들어 레바우디오시드 A, 글리시르히진 등)) 및 합성 향미제(사카린, 아스파르탐 등)를 유리하게 사용할 수 있다. 상기 천연 탄수화물의 비율은 본 발명의 조성물 100 ㎖ 당 일반적으로 약 1~20 g, 바람직하게는 약 5~12 g이다.The health functional beverage composition of the present invention is not particularly limited to other ingredients except for containing the extract as an essential ingredient in the indicated proportions, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. 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. have. The proportion of the natural carbohydrate is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.
상기 외에 본 발명의 조성물은 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있다. 그밖에 본 발명의 조성물들은 천연 과일 쥬스 및 과일 쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율은 그렇게 중요하진 않지만 본 발명의 조성물 100 중량부 당 0 내지 약 20 중량부의 범위에서 선택되는 것이 일반적이다. 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 from the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.
또한, 본 발명의 추출물은 비만증의 예방 효과를 목적으로 식품 또는 음료에 첨가될 수 있다. 이 때, 식품 또는 음료 중의 상기 추출물의 양은 전체 식품 중량의 0.01 내지 15 중량%로 가할 수 있으며, 건강 음료 조성물은 100 ㎖을 기준으로 0.02 내지 5 g, 바람직하게는 0.3 내지 1 g의 비율로 가할 수 있다.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.
본 발명의 발효 또는 미발효된 금은화, 진피의 혼합생약 추출물은 마우스 배아 섬유아세포(mouse embryo fibroblast) 유래 지방세포인 3T3-L1에서 지방분화를 억제시켜주는 효과와 고지방 식이로 비만을 유도한 흰쥐에서 체중감소 및 체지방 감소, 혈중지질농도 감소뿐만 아니라 렙틴(leptin), 아디포넥틴(adiponectin)의 혈중 농도 감소효과를 발휘하므로 비만 예방 및 치료를 위한 약학조성물, 건강기능식품 및 건강보조식품에 유용하게 이용될 수 있다.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.
도 1은 지방세포의 cell line인 3T3-L1의 MTT assay에 대한 F-CL의 효과를 나타낸 도이며,1 is a diagram showing the effect of F-CL on MTT assay of 3T3-L1, a cell line of adipocytes,
도 2는 지방세포의 cell line인 3T3-L1의 Oil Red-O staining에 대한 F-CL의 효과를 나타낸 도이며,2 is a diagram showing the effect of F-CL on Oil Red-O staining of 3T3-L1, a cell line of adipocytes,
도 3은 한약재 추출물이 AMPK와 ACC 활성에 미치는 영향에 대한CL과 F-CL의 효과를 나타낸 도이며, 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,
도 4는 고지방식이 투여로 인한 비만쥐의 몸무게에 대한 F-CL의 효과를 나타낸 도이며, Figure 4 is a diagram showing the effect of F-CL on the weight of obese mice due to high-fat diet administration,
도 5는 고지방식이 투여로 인한 비만쥐의 간무게에 대한 F-CL의 효과를 나타낸 도이며, 5 is a diagram showing the effect of F-CL on the liver weight of obese mice due to high-fat diet administration,
도 6(a-d)은 고지방식이 투여로 인한 비만쥐의 체지방 무게에 대한 F-CL의 효과를 나타낸 도이며,Figure 6 (a-d) is a diagram showing the effect of F-CL on the body fat weight of obese mice due to high-fat diet administration,
도 7(a-c)은 고지방식이 투여로 인한 비만쥐의 혈중 지질농도에 대한 F-CL의 효과를 나타낸 도이며,Figure 7 (a-c) is a diagram showing the effect of F-CL on the blood lipid concentration of obese mice due to high-fat diet administration,
도 8(d-f)은 고지방식이 투여로 인한 비만쥐의 혈중 지질농도에 대한 F-CL의 효과를 나타낸 도이며,Figure 8 (d-f) is a diagram showing the effect of F-CL on the blood lipid concentration of obese mice due to high-fat diet administration,
도 9은 고지방식이 투여로 인한 비만쥐의혈중 Adiponectin, Leptin 농도에 대한 F-CL의 효과를 나타낸 도이며, 9 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,
도 10는 고지방식이 투여로 인한 비만쥐의 조직에서 AMPK와 ACC 활성도에 대한 F-CL의 효과를 나타낸 도이다.10 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.
하기에 본 발명의 추출물을 포함하는 조성물의 제제예를 설명하나, 본 발명은 이를 한정하고자 함이 아닌 단지 구체적으로 설명하고자 함이다.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.
제제예 1. 산제의 제조Formulation Example 1 Preparation of Powder
금은화, 진피 혼합 생약 추출물(F-CL) 20 mgGold Silver Cod, Dermis Mixed Herbal Extract (F-CL) 20 mg
유당 100 mg Lactose 100 mg
탈크 10 mg Talc 10 mg
상기의 성분들을 혼합하고 기밀포에 충진하여 산제를 제조한다.The above ingredients are mixed and filled in an airtight cloth to prepare a powder.
제제예 2. 정제의 제조Formulation Example 2 Preparation of Tablet
금은화, 진피 혼합 생약 추출물(F-CL) 10 mgGold Silver Cod, Dermal Mixed Herbal Extract (F-CL) 10 mg
옥수수전분 100 mg Corn starch 100 mg
유당 100 mg Lactose 100 mg
스테아린산 마그네슘 2 mg2 mg magnesium stearate
상기의 성분들을 혼합한 후 통상의 정제의 제조방법에 따라서 타정하여 정제를 제조한다.After mixing the above components, tablets are prepared by tableting according to a conventional method for preparing tablets.
제제예 3. 캅셀제의 제조 Formulation Example 3 Preparation of Capsule
금은화, 진피 혼합 생약 추출물(CL) 10 mgGold Silver Cod, Dermis Mixed Herbal Extract (CL) 10 mg
결정성 셀룰로오스 3 mg3 mg of crystalline cellulose
락토오스 14.8 mgLactose 14.8 mg
마그네슘 스테아레이트 0.2 mgMagnesium Stearate 0.2 mg
통상의 캡슐제 제조방법에 따라 상기의 성분을 혼합하고 젤라틴 캡슐에 충전하여 캡슐제를 제조한다.According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.
제제예 4. 주사제의 제조Formulation Example 4 Preparation of Injection
금은화, 진피 혼합 생약 추출물(CL) 10 mgGold Silver Cod, Dermis Mixed Herbal Extract (CL) 10 mg
만니톨 180 mgMannitol 180 mg
주사용 멸균 증류수 2974 mgSterile distilled water for injection 2974 mg
Na2HPO4,12H2O 26 mgNa 2 HPO 4 , 12H2O 26 mg
통상의 주사제의 제조방법에 따라 1 앰플당(2 ㎖) 상기의 성분 함량으로 제조한다.According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).
제제예 5. 액제의 제조Formulation Example 5 Preparation of Liquid
금은화, 진피 혼합 생약 추출물(F-CL) 20 mgGold Silver Cod, Dermis Mixed Herbal Extract (F-CL) 20 mg
이성화당 10 g10 g of isomerized sugar
만니톨 5 g5 g of mannitol
정제수 적량Purified water
통상의 액제의 제조방법에 따라 정제수에 각각의 성분을 가하여 용해시키고 레몬향을 적량 가한 다음 상기의 성분을 혼합한 다음 정제수를 가하여 전체를 정제수를 가하여 전체 100 ㎖으로 조절한 후 갈색병에 충진하여 멸균시켜 액제를 제조한다.According to the conventional method of preparing a liquid solution, each component is added to the purified water to dissolve it, the lemon flavor is added appropriately, the above components are mixed, purified water is added, the whole is adjusted to 100 ml by the addition of purified water, and then filled in a brown bottle. The solution is prepared by sterilization.
제제예 6. 건강 식품의 제조Formulation Example 6 Preparation of Healthy Food
금은화, 진피 혼합 생약 추출물(F-CL) 1000 ㎎ Gold Silver Cod, Dermal Mixed Herbal Extract (F-CL) 1000 mg
비타민 혼합물 적량Vitamin mixture proper amount
비타민 A 아세테이트 70 ㎍ 70 μg of Vitamin A Acetate
비타민 E 1.0 ㎎ Vitamin E 1.0 mg
비타민 B1 0.13 ㎎ Vitamin B1 0.13 mg
비타민 B2 0.15 ㎎ Vitamin B2 0.15 mg
비타민 B6 0.5 ㎎ Vitamin B6 0.5 mg
비타민 B12 0.2 ㎍0.2 μg of vitamin B12
비타민 C 10 ㎎ Vitamin C 10 mg
비오틴 10 ㎍10 μg biotin
니코틴산아미드 1.7 ㎎ Nicotinic Acid 1.7 mg
엽산 50 ㎍ Folate 50 ㎍
판토텐산 칼슘 0.5 ㎎ Calcium Pantothenate 0.5mg
무기질 혼합물 적량Mineral mixture
황산제1철 1.75 ㎎ Ferrous Sulfate 1.75 mg
산화아연 0.82 ㎎ Zinc Oxide 0.82 mg
탄산마그네슘 25.3 ㎎ Magnesium carbonate 25.3 mg
제1인산칼륨 15 ㎎ Potassium monophosphate 15 mg
제2인산칼슘 55 ㎎ Dibasic calcium phosphate 55 mg
구연산칼륨 90 ㎎ Potassium Citrate 90 mg
탄산칼슘 100 ㎎ Calcium Carbonate 100 mg
염화마그네슘 24.8 ㎎ Magnesium chloride 24.8 mg
상기의 비타민 및 미네랄 혼합물의 조성비는 비교적 건강식품에 적합한 성분을 바람직한 실시예로 혼합 조성하였지만, 그 배합비를 임의로 변형 실시하여도 무방하며, 통상의 건강식품 제조방법에 따라 상기의 성분을 혼합한 다음, 과립을 제조하고, 통상의 방법에 따라 건강식품 조성물 제조에 사용할 수 있다.Although the composition ratio of the above-mentioned vitamin and mineral mixtures is mixed with a component suitable for a health food in a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method. The granules may be prepared and used for preparing a health food composition according to a conventional method.
제제예 7. 건강 음료의 제조Formulation Example 7 Preparation of Healthy Drink
금은화, 진피 혼합 생약 추출물(CL) 1000㎎ Gold silver coin, dermis mixed herbal extract (CL) 1000mg
구연산 1000 ㎎ Citric acid 1000 mg
올리고당 100 g100 g oligosaccharides
매실농축액 2 gPlum concentrate 2 g
타우린 1 g1 g of taurine
정제수를 가하여 전체 900 ㎖Add 900 ml of purified water
통상의 건강음료 제조방법에 따라 상기의 성분을 혼합한 다음, 약 1시간 동안 85℃에서 교반 가열한 후, 만들어진 용액을 여과하여 멸균된 2ℓ 용기에 취득하여 밀봉 멸균한 뒤 냉장 보관한 다음 본 발명의 건강음료 조성물 제조에 사용한다. After mixing the above components in accordance with a conventional healthy beverage production method, and then stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilized and then refrigerated and stored in the present invention For the preparation of healthy beverage compositions.
상기 조성비는 비교적 기호음료에 적합한 성분을 바람직한 실시예로 혼합 조성하였지만, 수요계층, 수요국가, 사용용도 등 지역적, 민족적 기호도에 따라서 그 배합비를 임의로 변형 실시하여도 무방하다.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.
이하, 본 발명을 하기 실시예 및 실험예에 의해 상세히 설명한다.Hereinafter, the present invention will be described in detail by the following Examples and Experimental Examples.
단, 하기 실시예 및 실험예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예, 참고예 및 실험예에 의해 한정되는 것은 아니다.However, the following Examples and Experimental Examples are only illustrative of the present invention, the contents of the present invention is not limited by the following Examples, Reference Examples and Experimental Examples.
실시예 1. 금은화, 및 진피 생약 발효 및 추출물 제조Example 1 Fermentation and Extract Preparation of Gold Coin and Dermis Herbal Medicine
1-1. 금은화 발효물의 제조1-1. Preparation of Gold Silver Fermentation
수분 함수율이 10% 미만으로 건조된 금은화(구입처 신진물산)를 15 kg을 준비 및 선별하고 120℃에서 20분간 멸균기(CT-DAC 80 및 코아테크)로 멸균과정을 거친 후에, 발효균주(Aspergillus oryzae, 충무발효)를 시료 중량의 10%를 첨가하여 수분 30%의 조건에서 온도 30℃에서 10일간 발효장치(온도제어 발효실, (주)이조바이오)에서 발효를 수행하여 금은화 발효물을 수득하였다. 발효가 완료된 발효물을 57℃에서 48시간 동안 건조시킨 후에 덩어리진 입자를 손으로 가볍게 비벼 자체 분리되도록 풀어주고 미세입자를 분리 및 선별하였다.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) , Fermented fermented radish) was added to 10% of the weight of the sample and fermented 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 a 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 freely separate themselves, and the fine particles are separated and selected.
이후에 180℃에서 원적외선 초제로 20분간 덖음과정을 수행하여 금은화 고체 발효물 13 Kg을 수득하여 하기에 사용하였다.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. 진피 발효물의 제조1-2. Preparation of Dermal Fermentation Products
수분 함수율이 10% 미만으로 건조된 진피(신진물산) 18 kg을 준비 및 분쇄하고 각 원료별 입자크기가 0.2-0.5 mm가 되도록 별하고 120℃에서 20분간 멸균기(CT-DAC 80, 코아테크)로 멸균과정을 거친 후에, 발효균주(Aspergillus oryzae, 충무발효)를 시료 중량의 10%를 첨가하여 수분 30%의 조건에서 온도 30℃에서 10일간 발효장치(온도제어발효실 (주)이조바이오)에서 발효를 수행하여 금은화 발효물을 수득하였다. 발효가 완료된 발효물을 57℃에서 48시간 동안 건조시킨 후에 덩어리진 입자를 손으로 가볍게 비벼 자체 분리되도록 풀어주고 미세입자를 분리 및 선별하였다.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 in a fermentation apparatus (Izobio Co., Ltd.) for 10 days at 30 ° C under 30% moisture. The fermentation was carried out to obtain the goldsmith fermentation. After fermentation is completed, the fermented product is dried at 57 ° C. for 48 hours, the agglomerated particles are lightly rubbed by hand to freely separate themselves, and the fine particles are separated and selected.
이후에 180℃에서 원적외선 초제로 20분간 덖음과정을 수행하여 진피 고체 발효물 15 Kg을 수득하여 하기에 사용하였다.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. 발효배합 추출물의 제조1-3. Preparation of Fermentation Blend Extract
위의 공정으로 발효된 금은화, 진피, 미발효된 금은화, 진피를 3:2 중량비로 5 kg 측정한 후 10배의 증류수를 가수한 후 95℃로 상승 후 12시간 추출하였다. 추출 후 침전물을 거름종이(Advantec, 71101903)를 이용하여 여과한 다음 이 여과액을 회전 진공증류기(EYELA, A-1000s)에서 감압 농축 하였다. 상기 농축액을 -70℃ 저온 냉동기(deep freezer)에서 12시간 방치하고, 72시간 동안 냉동 건조기(freeze dryer)로 건조하여 발효된 혼합추출물 분말 2.1 kg(수득율 42%)과 상기 발효과정을 수행하지 않은 재료로 제조한 미발효 혼합 추출물 분말 1.6 kg(수득율 32%)을 수득하였다(이하 발효된 혼합추출물을 'F-CL', 미발효 혼합 추출물 'CL'이라 명명함). Fermented gold silver, dermis, unfermented gold silver and dermis fermented by the above process was measured 5 kg in a 3: 2 weight ratio, and then distilled water of 10 times and then heated to 95 ℃ and extracted for 12 hours. After extraction, the precipitate was filtered using a filter paper (Advantec, 71101903), and the filtrate was concentrated under reduced pressure in a rotary vacuum distillation machine (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 fermented mixed extract 'CL').
참고예 1. 실험재료의 준비Reference Example 1. Preparation of Experimental Materials
3T3-L1 마우스유래 배아섬유아세포는 미국세포주은행(American Type Culture Collection; ATCC, CL-173TM)에서 구입하였으며, Dulbeccos modified Eagles media (DMEM, SH30243.01), Bovine calf serum (BCS, SH30401.01), Fetal bovine serum (FBS, SH30396.03) 및 페니실린과 스트렙토마이신(penicillin/streptomycin; PS)은 하이클론(SV30010, Hyclone, USA)에서 구입하였다. 인슐린(Insulin, 090-03446)과 디메칠술폭사이드(dimethyl sulfoxide; DMSO, D2650), 오일레드오(Oil Red-O, O0625-25), 덱사메타손(dexamethasone (DEX, D4902-100MG)과 이소부칠메틸잔틴(isobutylmethylxanthine (IBMX, I5879-250MG)는 시그마-알드리치(Sigma-Aldrich company, St. Louis, MO, USA)에서 구입하여 사용하였다.3T3-L1 mouse-derived embryonic fibroblasts were purchased from the 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.
참고예 2. 실험동물의 준비Reference Example 2. Preparation of Laboratory Animals
실험동물은 체중 28~32 g의 생후 5주령의 ICR mice(오리엔트 사)를 사용하였고, 대구한의대학교 동물 사육실에서 일정한 조건(온도: 21±2, 명암: 12시간 명암주기)하에, 사료와 음수의 자유로운 섭취가 가능하도록 하였으며, 실험시작 전까지 물과 먹이를 충분히 제공하였다.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.
참고예 3. 3T3-L1 세포배양 Reference Example 3 3T3-L1 Cell Culture
3T3-L1 마우스유래 배아섬유아세포(mouse embryo fibroblast)를 하기와 같은 실험방법으로 배양하였다(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). 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).
먼저, 10% 우태아 혈청(bovine calf serum)을 함유하는 Dulbecco's modified Eagle's medium(DMEM)로 37℃, 5% CO2 incubator에서 배양하였다(Day 0). 8일 동안 배양한 다음에 전지방세포(preadipocyte)를 10% fetal bovine serum(FBS)을 함유한 DMEM 배지(167 nM insulin, 0.5μΜ isobutylmethylxanthine, 1μΜ dexamethasone 첨가)로 옮겨 2일간 배양하였다(Day 2). 167 nM 인슐린이 있는 배지에서 2일간 더 배양한 다음에 10% FBS/DMEM 배지에서 4일 동안 배양하여 성숙한 지방세포(adipocytes)로 만들었다. First, incubated in Dulbecco's modified Eagle's medium (DMEM) containing 10% bovine calf serum (DMEM) at 37 ℃, 5% CO 2 incubator (Day 0). After 8 days of incubation, the preadipocytes were transferred to DMEM medium containing 10% fetal bovine serum (FBS) (167 nM insulin, 0.5 μΜ isobutylmethylxanthine, 1 μΜ dexamethasone added) and incubated for 2 days (Day 2). . Two more days of culture in 167 nM insulin were followed by 4 days in 10% FBS / DMEM medium to make mature adipocytes.
참고예 4. 발효 금은화, 발효 진피 혼합 생약 추출물의 투여 및 비만 유도 Reference Example 4 Administration of Fermented Gold Silver Coated Fermented Dermal Mixed Herbal Extract and Induction of Obesity
비만을 유발하기 위하여 문헌에 기재된 식이조성을 이용하여 하기와 같이 실험을 수행하였다(Lee J. S. 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). 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).
먼저, 정상군을 제외한 실험군에는 AIN-76 조성을 기초로 한 식이에 표 1과 같이 콜레스테롤 1%와 Lard 15%를 첨가한 펠렛 형태의 고형사료(#102038, AIN-76A Based High Fat/High Carb Purified Rodent Diet with Cholesterol & Cholic Acid, Dyet, USA) 6주간 먹였으며, 실험동물은 각 군당 15마리씩 배정하였다. 즉, 정상군(Normal), 고지방대조군(HF), 고지방사료 미발효 혼합추출물(CL-5%), 고지방사료 발효 혼합추출물(F-CL-5%)로 나누고, 시료군에는 상기 실시예 1에서 수득한 동결 건조한 혼합 생약 추출물 분말을 고지방식이에 5% 첨가하여 매일 오전에 물과 함께 공급하였으며, 매일 20 g의 사료를 할당하였다.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.
표 1
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%(수율32%) - - 16 - -
F- CL : 5%(수율42%) - - - 21 -
Garcinia cambogia 1%(HCA 0.6%) - - - - 101)
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
Table 1
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
참고예 5. 통계처리Reference Example 5. Statistics Processing
실험성적의 통계적 유의성은 SPSS 프로그램(Version 14.01)의 투웨이(Two-way) ANOVA와 LSD 사후검정을 이용하여, P<0.05 인 경우에 유의한 것으로 인정하였으며, 정상군과의 통계학적 유의성(#) 및 고지방식이투여군(HF)과의 통계학적 유의성(*)을 도면 및 표에 각각 표기하였다. The statistical significance of the experimental results was found to be significant in the case of P <0.05 using two-way ANOVA and LSD post-test of the SPSS program (Version 14.01), and statistical significance with the normal group (#) And statistical significance (*) with the high fat diet group (HF) are shown in the figures and tables, respectively.
실험예 1. 세포독성(MTT assay)Experimental Example 1. Cytotoxicity (MTT assay)
발효, 미발효, 혼합 추출물이 3T3-L1 세포에 미치는 영향을 알아보기 위하여 MTT검정법(methylthiazol tetrazolium bromide)을 이용하여 측정하였다(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). 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).
MTT 검정법은 살아있는 세포의 미토콘드리아에 있는 탈수소효소에 의해 생성된 blue formazan 결정을 DMSO(dimethyl sulfoxide)로 녹여서 발색시켜 그 흡광도를 측정하는 방법이다. 참고예 1에서 배양한 3T3-L1 cell line을 사용하였으며, 실험에는 지수적 성장기에 있는 세포를 사용하셨다. 즉, 5 X 104 cells/ml을 일정기간 아무처치 없이 동량의 5% tryphan으로 잘 섞어 살아있는 세포수를 측정하였고, 생존능 실험은 모두 3개 well의 평균가를 이용하여 평가하였다. Well당 MTT 용액(2 mg/ml)을 50 ㎕씩 넣어 37℃, 5% CO2 항온기에서 2시간 반응시킨 후 배지는 제거하고 DMSO를 well당 100 ㎕씩 넣어 5분간 plate shaker에서 blue formazan 결정을 용해시킨 다음 ELISA reader(Techan, Germany) 540 nm에서 흡광도를 측정하였다.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 the cells in exponential growth phase were used for the experiment. In other words, 5 × 10 4 cells / ml was mixed well with the same amount of 5% tryphan without any treatment for a certain period of time, and the number of living cells was measured. The viability test was evaluated using the average value of all 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).
그 결과, 도 1에 나타난 바와 같이 발효 한약재(F-CL)와 미발효 한약재(CL)를 농도별(0.1, 0.3, 1 mg/㎖)로 24시간 처리하여 MTT assay를 실험한 결과, 대조군에 비해 시료 농도에 의존적으로 지방세포의 생존율이 감소하는 것으로 관찰되었으며, 특히 발효 한약재(F-CL) 0.1 mg/㎖ 농도에서 미발효 한약재 추출물보다 유의하게 세포생존율이 저해되었다.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).
실험예 2. 세포를 이용한 Experimental Example 2. Using Cells oil red-Ooil red-O 염색 dyeing
3T3-L1 세포에서 지방세포 분화 및 지방 생성에 발효, 미발효, 혼합 추출물이 미치는 영향을 알아보기 위하여 Oil Red-O 염색 실험을 수행하였다(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 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 염색법은 분화된 3T3-L1 세포에 Oil Red-O 시약으로 염색하여 세포내 생성된 지방을 측정하는 방법이다. 분화된 세포의 배지를 제거하고 D-PBS(DPBS, SH30028.03)로 세포를 세척하고 pH 7.2 Cacodylate buffer로 2시간 고정하고 Oil-red O Solution으로 염색하였다. 세포 염색이 끝난 후 40% Isopropyl alcohol(2-Propanol,67-63-0)로 3번 세척 후 건조하여 세포 내 지방의 크기를 광학현미경(AE 31, Motic, USA)으로 관찰하였다. Oil Red-O staining is a method of measuring the amount of intracellular fat produced by staining 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).
실험결과, 도 2에 나타나는 바와 같이, 3T3-L1 세포의 분화과정 중에 발효, 미발효 혼합 추출물을 각각 100 ㎍/㎖ (B), 150 ㎍/㎖ (C), 200 ㎍/㎖ (D)의 농도로 시료를 처리한 결과, 대조군(control)에 비해서 농도 의존적으로 세포분화 및 지방생성을 억제하였으며, 각각의 한약재 추출물을 현미경 및 육안으로 관찰한 결과, 발효 혼합 추출물 처리군이 미발효 혼합 추출물보다 지방축적 저해효과가 뛰어났다. 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), and as a result of observing each herbal extract microscopically and visually, the fermented mixed extract treatment group was higher than the unfermented mixed extract. It was excellent in inhibiting fat accumulation.
실험예 3. 3T3-L1 Cell WesternExperimental Example 3. 3T3-L1 Cell Western
발효 혼합 추출물이 AMPK, ACC와 PPAR-γ 활성에 미치는 영향을 알보기 위하여 3T3-L1 Cell Western을 수행하였다(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).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).
농도별로 처리된 3T3-L1 cell을 D-PBS(DPBS, SH30028.03)로 washing 한 후 1mM EDTA가 포함된 50 mM tris-HCl(pH7.5) buffer 2 ml를 첨가하여 세포를 떼어낸 후 Branson model 185 sonifier를 이용하여 40 watt에서 5초간 sonication하였다. 13,000 rpm 4℃에서 5분간 원심분리한 후 20 ㎍의 동일한 양의 단백질을 denature시키고 8% 또는 10% SDS-PAGE를 분리한 후, PVDF membrane으로 transfer시킨다. mebrane을 TBS-T로 용해한 5% skin milk로 2시간 동안 blocking 한다음, rabbit polyclonal anti-PPARγ, AMPK, ACC antibody로 1시간 동안 처리하였다. TBS-T로 세척한 후에 horseradish peroxidase인 2차 항체로 1시간 동안 처리하여 다시 TBS-T로 washing 한 후 ECL detection 시약으로 immunorective bands를 관찰하였다.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.
실험결과, 도 3에 나타나는 바와 같이. 발효 혼합추출물에서 AMPK의 인산화 활성이 증가한 것으로 나타났다. 그에 비해 미발효 혼합 추출물에서는 AMPK의 인산화 활성에 아무런 영향을 미치지 않는 것으로 확인되었다. 또한 AMPK의 인산화 활성은 발효 혼합 추출물의 농도에 비례하여 증가하는 양상을 보였다. AMPK 인산화 활성에 따른 ACC 마찬가지로 발효 혼합 추출물에서 유의하게 활성화된 것으로 관찰되었다. 뿐만 아니라 PPAR-γ는 지방세포에 나타나는 특이적인 전사 인자로 분화 후기에 발현되며 여러 호르몬인 adipocytokine과 adipogenic protein 발현에 중요한 역할을 한다. 발효 혼합 추출물과 미발효 혼합 추출물에서 PPAR-γ 단백질 발현 억제활성을 확인한 결과 발효 추출물에 PPAR-γ가 매우 감소하는 양상을 보였고, 미발효 혼합 추출물에서는 농도에 따른 차이가 유의하게 나타나지 않았다.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.
실험예 4. 고지방 식이를 이용한 비만 유도 쥐의 식이효율, 체중측정 및 조직의 채취Experimental Example 4. Dietary efficiency, weight measurement and tissue collection in obese rats using high fat diet
식이 섭취량은 매일 오전에 측정하여, 일일 먹이 섭취량을 계산하였으며, 체중은 일주일에 한 번씩 측정하였다. 식이효율(FER; Food efficiency ratio)은 실험기간 동안의 체중 증가량을 구하고 그 값을 식이섭취량으로 나눈 값이다(FER=body weight gain/food intake). 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 weight gain during the test period and the value divided by the dietary intake (FER = body weight gain / food intake).
상기 실험 결과 표 2 및 도 4에 나타나는 바와 같이 6주 후 정상군(NOR)의 체중보다 고지방식이 투여군(HF)에서 유의성 있게 체중이 증가하였으며, HCA, CL, F-CL군에서는 체중의 증가를 유의적으로 억제하는 결과를 얻을 수 있었다. 식이섭취량을 보면 정상군(NOR)과 비교하였을 때, HCA군와 CL, F-CL군에서 유의성 있는 감소를 보였다. 체중의 변화율에 있어서는 고지방식이 투여군(HF)에 비해 정상군(NOR)과 HCA, F-CL군에서 유의적인 감소를 보였다. 식이효율(FER)에 있어서도 정상군(NOR)에 비해 HF군, HCA, CL, F-CL군에서 유의적으로 높은 결과를 보였다.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.
한편, 실험동물은 희생 전 15시간 절식 시킨 후 CO2로 마취한 상태에서 Liver 및 부위별 내장지방조직(부고환;epididymal, 내장;visceral adipose tissue)과 갈색지방(brown adipose tissue; BAT)을 채취하였다. 조직 염색용 조직은 포르말린 용액에 고정 시켰으며, 분석용 체지방조직은 액체 질소로 급속 냉동시켜 사용하기 전까지 -80℃에 보관하였다.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.
상기 실험 결과 도 5 및 도 6(a-d)에 의하면 정상군에 비해 고지방 식이 투여군(HF)군에서 간 무게, 부고환 지방 무게, 신장주위 지방 무게, 내장 무게, 갈색지방(BAT)가 모두 유의하게 증가하였으며, HCA, CL, F-CL군에서 간장무게, 부고환지방 무게, 신장주위 지방조직 무게 및 갈색지방 무게가 감소되었으며, 그 중에서도 F-CL군에서 유의하게 억제되었다. 이 결과는 비만에 있어서 단순한 체중감소 보다는 심혈관계 질환과 같은 합병증을 야기시키는 것으로 알려진 체지방 감소에 대한 강력한 F-CL의 효과를 보여주는 것으로서 비만 치료제로서의 더 큰 의미가 있다고 할 수 있다.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.
표 2
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#
TABLE 2
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 #
실험예 5. 비만을 유발시킨 쥐의 채혈 및 혈청 분석Experimental Example 5. Blood collection and serum analysis of obese rats
각 군의 실험동물을 희생 전 15시간 절식시킨 후 CO2로 가볍게 마취한 다음, 복부 대정맥에서 혈액을 채취하였다. 채취한 혈액은 test tube에 넣고 상온에서 1시간 방치한 다음 4℃, 3,500 rpm에서 15분간 원심분리 하여 혈청을 분리하고, 혈청 중 총콜레스테롤(total cholesterol; TC)은 C. Allain 1의 효소법(Allain C. et al., Enzymatic determination of total serum cholesterol, Clin. Chem. 20(4), pp.470-475, 1974)에 따라 분석시약(L-type GHO M, Wako, 일본)을 사용하였고 중성지방(triglyceride) 함량은 Van Handel등의 효소법(Van Handel E. et al, Micromethod for the direct determination of serum triglycerides, J. Lab. Clin. Med. 50(1), pp.152-157, 1957)에 의하여 검사시약(L-type TG M, Wako, 일본)을 사용하였다. 고밀도지단백(HDL-cholesterol)과 저밀도지단백(LDL-cholesterol) 농도는 Warnick의 효소법(Warnick G. R. et al., Dextran sulfate-Mg2+ precipitation procedure for quantitationof high density lipoprotein cholesterol, Clin. Chem. 20(6), pp.1397-1388, 1982)에 의하여 검사시약(Choesterol N HDL, Choesterol N LDL, SEKISUI, 일본)으로 측정하였다. 간 기능과 관련된 트랜스아미나아제(transaminase)인 혈청 중 에이엘티(ALT; Alanine aminotransferase)와 혈청 에이에스티(AST; Aspartate aminotransferase) 함량은 분광광도법(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)을 이용한 검사시약(L-type ALT J2, L-type AST J2, Wako, 일본)을 사용하여 자동화된 생화학 분석기(Hitachi 7080, Japan)로 분석하였다. 혈청 중 렙틴(leptin)의 농도는 Leptin ELISA kit(900-015, Assay Designs, U.S.A.), 혈청 중 아디포넥틴(adiponectin)의 농도는 Adiponectin ELISA kit(44-ADPR-0434, ALPCO Diagnostics, U.S.A.)를 사용하여 ELISA reader(Tecan, Germany)로 분석하였다. After the animals in each group were sacrificed 15 hours fasting before the lightly anesthetized with CO 2 was collected, and then, the blood in 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 measured using Adiponectin ELISA kit (44-ADPR-0434, ALPCO Diagnostics, USA). Analysis was performed with an ELISA reader (Tecan, Germany).
실험결과, 도 7(a-c) 및 도 8(e-f)에 나타나는 바와 같이, 정상군에 비해 고지방 식이 투여군(HF)군에서 혈청 중 ALT, AST와 중성지방 농도 및 저밀도지단백(LDL) 함량이 증가하였으나, F-CL의 투여로 혈청 중 ALT, AST와 중성지방 농도 및 저밀도지단백(LDL) 함량이 감소되었으며, 혈청 중 고밀도지단백(HDL) 함량은 정상군에 비해 고지방 식이 투여군(HF)군에서 감소하였으나, F-CL 투여로 고밀도지단백(HDL) 함량이 증가하는 경향을 보였다.As shown in FIG. 7 (ac) and FIG. 8 (ef), ALT, AST and triglyceride concentrations and low density lipoprotein (LDL) content in serum were increased in the high fat diet group (HF) group compared to the normal group. ALT, AST and triglyceride concentrations and low density lipoprotein (LDL) content in serum were decreased by administration of F-CL, and high density lipoprotein (HDL) content in serum was decreased in high fat diet group (HF) group compared to normal group. , F-CL administration tended to increase HDL content.
인슐린감수성 호르몬으로 작용하면서 지방세포에서 분비되는 아디포넥틴(adiponectin)의 혈청 중 함량은 도 9-a에 나타나는 바와 같이, 정상군(NOR)에서 고지방 식이 투여군(HF)보다 증가하였으며, F-CL 투여로 혈청 중 아디포넥틴 (adiponectin) 함량은 고지방 식이 투여군(HF)보다 증가하는 경향을 보였다. As shown in Figure 9-a, the serum content of adiponectin secreted from adipocytes while acting as an insulin-sensitive hormone was increased in the normal group (NOR) than the high-fat diet group (HF), by F-CL administration. The adiponectin content in the serum tended to be higher than that in the high fat diet group (HF).
지방세포에서 분비되는 펩타이드로서, 식욕조절과 관련이 있다고 알려진 렙틴(leptin)의 혈청 중 함량은 도 9-b에 나타나는 바와 같이, 정상군(NOR)에 비해 고지방 식이 투여군(HF)에서 유의하게 증가하였으며, F-CL 투여로 혈청 중 렙틴(leptin) 함량은 고지방 식이 투여군(HF)보다 유의하게 감소하는 경향을 보였다. As a peptide secreted from adipocytes, the serum content of leptin, known to be related to appetite control, was significantly increased in the high fat diet administered group (HF) compared to the normal group (NOR), as shown in FIGS. Leptin content in serum was significantly lower than that of high fat diet group (HF) by F-CL administration.
실험예 6. 근육에서 AMPK ,ACC 및 PPAR-γ의 활성Experimental Example 6. Activity of AMPK, ACC and PPAR-γ in Muscle
발효 혼합 추출물이 AMPK, ACC와 PPAR-γ 활성에 미치는 영향을 알아보기 위하여 3T3-L1 Cell Western을 수행하였다(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).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).
조직을 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 Na3VO4, protease inhibitor cocktail)를 사용하여 균질화 시킨 다음 4℃에서 14,000 g에서 10분간 원심분리하여 상등액을 취한 다음 단백질 함량을 BCA reagent로 정량을 한 뒤, 20 ㎍을 8% SDS-PAGE에서 전기영동 시켰다. 전개시킨 gel을 PVDF membrane(Millipore)에 transfer한 후 5% bovine serum albumin(BSA)-TBST(TBS, 0.1% Tween-20)로 blocking 시켰다. 5% bovine serum albumin(BSA)-TBST에 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) primary antibody를 overnight으로 반응시키고, TBST용액으로 5분간 6회 washing 한후 secondary antibody(goat anti-rabbit-HRP, 1:5000 dilution, Assay designs)를 5% bovine serum albumin(BSA)-TBST에 넣어 1시간 동안 반응시킨 다음 TBST용액으로 5분간 6회 washing하였다. 이후 ECL reagent(Amersham Pharmacia Biotech., Arlington Heights, IL, USA)를 사용하여 1분간 반응한 뒤 Image Quant software(Buckinghamshire, UK)를 사용하여 Membrane에 부착된 단백질을 확인하였다.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).
실험결과, 도 10에 나타난 바와 같이, 근육에서의 비만 관련을 AMPK, ACC 인산화에 미치는 발효 한약재 추출물과 미발효 한약재 추출의 효과를 Western blot으로 확인하였을때, 밴드의 발현정도가, P-AMPK에서 고지방 식이 대조군(HF)과 비교 하였을때, 발효 한약재추출물(F-CL)에서 활성이 증가한 것으로 나타났다. P-AMPK와 마찬가지로 P-ACC도 고지방식이군(HF)과 비교 하였을때 F-CL군에서 활성화 되었고 약물대조군(HCA) 보다 유의하게 활성화된 것으로 관찰되었다.As a result, as shown in Figure 10, when the effects of the fermented herbal extracts and unfermented herbal extracts on the AMPK, ACC phosphorylation in muscle related to obesity in the muscle was 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).
본 발명의 발효 또는 미발효된 금은화, 진피의 혼합생약 추출물은 마우스 배아 섬유아세포(mouse embryo fibroblast) 유래 지방세포인 3T3-L1에서 지방분화를 억제시켜주는 효과와 고지방 식이로 비만을 유도한 흰쥐에서 체중감소 및 체지방 감소, 혈중지질농도 감소뿐만 아니라 렙틴(leptin), 아디포넥틴(adiponectin)의 혈중 농도 감소효과를 발휘하므로 비만 예방 및 치료를 위한 약학조성물, 건강기능식품 및 건강보조식품에 유용하게 이용될 수 있다.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.

Claims (10)

  1. 발효 또는 미발효된 금은화 및 진피 혼합생약 추출물을 유효성분으로 함유하는 비만증의 예방 및 치료용 약학조성물.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.
  2. 제 1항에 있어서,The method of claim 1,
    상기 금은화 : 진피의 중량 혼합비(w/w)가 1-5 : 1(w/w)로 배합된 배합물임을 특징으로 하는 약학조성물.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).
  3. 제 1항에 있어서,The method of claim 1,
    상기 혼합 생약 추출물은 물, 탄소수 1 내지 4의 저급알코올 또는 이들의 혼합용매로부터 선택된 용매에 가용한 추출물임을 특징으로 하는 약학조성물.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.
  4. 제 1항에 있어서,The method of claim 1,
    상기 발효 추출물은 개개 생약을 수분 함수율이 20% 미만으로 건조시킨 시료를 80 내지 150℃에서 10분 내지 70분간 멸균과정을 거치는 멸균단계; 멸균된 시료에 발효균주를 시료 중량의 1 내지 30% 양으로 첨가하여 수분 10 내지 50%의 조건에서 10 내지 50℃ 온도에서 1일 내지 30일간 발효를 수행하는 제 2단계; 상기 발효가 완료된 발효물을 30 내지 100℃ 온도에서 12시간 내지 72시간 동안 건조시키는 제 3단계; 상기 건조된 덩어리진 입자를 비벼 자체 분리되도록 풀어주고 미세입자를 분리 및 선별하는 제 4단계; 상기 선별된 발효물을 100 내지 300℃에서 원적외선 초제로 5분 내지 60분간 덖음과정을 수행하는 제 5단계 공정을 포함하는 공정으로 수득됨을 특징으로 하는 약학조성물.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 ℃.
  5. 발효 또는 미발효된 금은화 및 진피 혼합생약 추출물을 유효성분으로 함유하는 비만증의 예방 및 개선용 건강기능식품.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.
  6. 제 5항에 있어서, 상기 건강기능식품의 형태는 분말, 과립, 정제, 캡슐, 환제, 티백제, 침출차 또는 건강 음료인 건강기능식품.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.
  7. 비만증 억제활성을 나타내는 발효 또는 비발효된 금은화 및 진피의 혼합 생약 추출물을 주성분으로 함유하는 건강보조식품.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.
  8. 금은화 및 진피 개개 생약을 수분 함수율이 20% 미만으로 건조시킨 시료를 80 내지 150℃에서 10분 내지 70분간 멸균과정을 거치는 멸균단계; 멸균된 시료에 발효균주를 시료 중량의 1 내지 30% 양으로 첨가하여 수분 10 내지 50%의 조건에서 10 내지 50℃ 온도에서 1일 내지 30일간 발효를 수행하는 제 2단계; 상기 발효가 완료된 발효물을 30 내지 100℃ 온도에서 12시간 내지 72시간 동안 건조시키는 제 3단계; 상기 건조된 덩어리진 입자를 비벼 자체 분리되도록 풀어주고 미세입자를 분리 및 선별하는 제 4단계; 상기 선별된 발효물을 100 내지 300℃에서 원적외선 초제로 5분 내지 60분간 덖음과정을 수행하는 제 5단계 공정을 수행하여 발효 시료를 얻는 단계; 상기 단계에서 얻은 발효 시료 또는 미발효된 건조생약재료인 금은화 및 진피을 세척하여, 금은화 : 진피의 중량 혼합비(w/w)가 1-5 : 1(w/w)로 배합하고 상기 시료의 1 내지 20배 (w/v) 중량의 물, 메탄올, 에탄올 또는 이들의 혼합용매로 50 내지 120에서 1시간 내지 5시간 동안 열수 추출법, 냉침 추출법 또는 초음파 추출법을 수행한 후에 얻어진 추출액을 여과지로 여과한 후에 얻어진 여과물을 동결건조, 상온건조 또는 열풍건조를 수행하여 제 1항의 발효 또는 미발효된 금은화 및 진피의 혼합 생약 추출물을 제조하는 제조방법.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: dermal weight mixing ratio (w / w) of 1-5: 1 (w / w) is formulated and 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. The obtained filtrate is subjected to lyophilization, room temperature drying, or hot air drying to produce a mixed herbal extract of fermented or unfermented sterling silver and dermis of claim 1.
  9. 발효 또는 미발효된 금은화 및 진피의 혼합 생약 추출물을 비만증 환자에게 투여함을 포함하는, 비만증 환자를 치료하기 위한 치료방법.A method for treating obese patients, comprising administering a fermented or unfermented fermented herb extract to the obese patient.
  10. 비만증 치료용 약제를 제조하기 위한 발효 또는 미발효된 금은화 및 진피의 혼합 생약 추출물의 용도.Use of a mixed herbal extract of fermented or unfermented sterling silver and dermis to prepare a medicament for the treatment of obesity.
PCT/KR2012/004237 2011-08-10 2012-05-30 Composition for the prevention and treatment of obesity containing an active ingredient in the form of a fermented or unfermented lonicera japonica and aurantii nobilis pericarpium mixed herbal-preparation extract, and a use therefor WO2013022178A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR1020110079661A KR101344189B1 (en) 2011-08-10 2011-08-10 A Composition comprising an extract of fermented or non-fermented Lonicerae Flos and Citri Reticulatae Pericarpium for treating or preventing obesity
KR10-2011-0079661 2011-08-10

Publications (1)

Publication Number Publication Date
WO2013022178A1 true WO2013022178A1 (en) 2013-02-14

Family

ID=47668657

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2012/004237 WO2013022178A1 (en) 2011-08-10 2012-05-30 Composition for the prevention and treatment of obesity containing an active ingredient in the form of a fermented or unfermented lonicera japonica and aurantii nobilis pericarpium mixed herbal-preparation extract, and a use therefor

Country Status (2)

Country Link
KR (1) KR101344189B1 (en)
WO (1) WO2013022178A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108478766A (en) * 2018-05-22 2018-09-04 北京未医堂健康科技有限公司 A kind of drug and preparation method thereof for treating internal moisture and microcirculation silting

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102087041B1 (en) * 2019-07-08 2020-03-10 (주)앤이바이오플러스 Manufacturing method of health food composition for diet

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20020016292A (en) * 2000-08-25 2002-03-04 박세혁 Tea produced with the fruit of the Ligustrum japonicum tree reduce weight and relieve constipation
KR20030071974A (en) * 2002-03-05 2003-09-13 심인섭 Pharmaceutical composition for medical treatment and prevention of obesity
KR20070103629A (en) * 2006-04-19 2007-10-24 김용민 Composition for restraint of obesity
KR20090025638A (en) * 2007-09-06 2009-03-11 주식회사 디에이치피코리아 Compositions for treatment and preventing obesity
KR20090043758A (en) * 2007-10-30 2009-05-07 바이오허브 주식회사 Composition for improvement of fatty liver

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20020016292A (en) * 2000-08-25 2002-03-04 박세혁 Tea produced with the fruit of the Ligustrum japonicum tree reduce weight and relieve constipation
KR20030071974A (en) * 2002-03-05 2003-09-13 심인섭 Pharmaceutical composition for medical treatment and prevention of obesity
KR20070103629A (en) * 2006-04-19 2007-10-24 김용민 Composition for restraint of obesity
KR20090025638A (en) * 2007-09-06 2009-03-11 주식회사 디에이치피코리아 Compositions for treatment and preventing obesity
KR20090043758A (en) * 2007-10-30 2009-05-07 바이오허브 주식회사 Composition for improvement of fatty liver

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
KWON, CHONG-SUK ET AL.: "Effect of Lonicerajaponica Flower on Body Weight Gain and Glucose Tolerance in Rodents.", FOOD SCI. BIOTECHNOL., vol. 13, no. 6, 2004, pages 768 - 771 *
YANG, GABSIK ET AL.: "Lipid Lowering Activity of Citri Unshii Pericarpium in Hyperlipemic Rats.", IMMUNOPHARMACOLOGY AND IMMUNOTOXICOLOGY., vol. 30, no. 4, 2008, pages 783 - 791 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108478766A (en) * 2018-05-22 2018-09-04 北京未医堂健康科技有限公司 A kind of drug and preparation method thereof for treating internal moisture and microcirculation silting

Also Published As

Publication number Publication date
KR101344189B1 (en) 2014-01-23
KR20130017305A (en) 2013-02-20

Similar Documents

Publication Publication Date Title
WO2012033378A2 (en) Composition comprising cudrania tricuspidata and coix lachryma-jobi having an obesity-suppressing activity and blood-sugar lowering effect, and a use therefor
WO2015002391A1 (en) Composition having a function for alleviating premenstrual syndrome and menstrual pain
WO2018174448A1 (en) Composition for treating and preventing climacteric syndrome containing combined herbal medicinal extract of white atractylis, mori fructus, chinese matrimony vine, longan, achyranthes, eucommia bark, and asparagus cochinchinensis merr. as active ingredient, and use of same
WO2018062777A1 (en) Muscular strength and muscle mass improving composition comprising soybean juice peptide derived from germinated soybean treated with unripened tangerine water
WO2013100340A1 (en) Composition comprising dendropanax morbiferus extract for improving male sexual function
WO2012134172A2 (en) Composition containing, as an active ingredient, an ethyl acetate fraction of schisandra chinensis baill, or wuweizisu c separated from the fraction, for preventing or treating obesity
WO2021080297A1 (en) Composition containing evening primrose flower extract as active ingredient for preventing or treating obesity or metabolic syndromes induced thereby
WO2020235828A1 (en) Composition for inhibiting osteoclasts containing agastache rugosa extract as active ingredient, and use thereof
KR101071044B1 (en) Composition comprising the mixed extract of Astragali radix, Poria and Phaseoli semen for preventing and treating obesity
WO2019050123A1 (en) Food composition for improving obesity or hyperlipidemia comprising terminalia chebula fruit extract and phyllanthus emblica extract
WO2013022178A1 (en) Composition for the prevention and treatment of obesity containing an active ingredient in the form of a fermented or unfermented lonicera japonica and aurantii nobilis pericarpium mixed herbal-preparation extract, and a use therefor
WO2013069934A1 (en) Composition for treating and preventing obesity, containing wheatgrass extract as active ingredient
WO2017082478A1 (en) Pharmaceutical composition for preventing or treating osteoporosis comprising soybean germinated embryo extract
WO2011074765A2 (en) Composition including fermented material for oriental medicine as an active ingredient for preventing and treating obesity or hyperlipidemia
WO2010008150A2 (en) Composition for the prevention or treatment of osteoporosis, containing a mixture of saururus chinensis and scutellaria baicalensis extracts as an active ingredient
WO2018221922A1 (en) Composition for preventing and treating muscle-related diseases, containing coptidis rhizoma extract, and use thereof
WO2021080298A1 (en) Composition containing enteroccocus faecalis as active ingredient for preventing or treating obesity or metabolic syndromes induced thereby
KR100769412B1 (en) A composition comprising an extract of pr-119 formulation for the prevention and treatment of benign prostatic hyperplasia
WO2017030419A1 (en) Composition including euphorbia supina raf. extract or fraction as active ingredient for preventing or treating obesity
WO2016003120A1 (en) Whitening composition comprising scutellaria alpina extract
KR101503834B1 (en) A composition comprising a crude extract or purified fraction extract of Plantago asiatica for treating or preventing hyperlipidemia and obesity
WO2015167240A1 (en) Composition containing scutellaria alpina extract
WO2015152653A1 (en) Composition comprising extract of alpine wormwood
WO2021045242A2 (en) Composition for ameliorating premenstrual syndrome symptoms, including chrysanthemum sibiricum extract
WO2016117762A1 (en) Composition containing gooseberry extract or glutathione

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 12821853

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 12821853

Country of ref document: EP

Kind code of ref document: A1