WO2014189176A1 - Ecklonia cava extract for reducing weight and process for preparation thereof - Google Patents

Ecklonia cava extract for reducing weight and process for preparation thereof Download PDF

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WO2014189176A1
WO2014189176A1 PCT/KR2013/007798 KR2013007798W WO2014189176A1 WO 2014189176 A1 WO2014189176 A1 WO 2014189176A1 KR 2013007798 W KR2013007798 W KR 2013007798W WO 2014189176 A1 WO2014189176 A1 WO 2014189176A1
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extract
ecklonia cava
present
ecklonia
enzyme
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PCT/KR2013/007798
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French (fr)
Korean (ko)
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권창주
윤인주
김인혜
강미혜
남택정
김아람
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주영엔에스(주)
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L17/00—Food-from-the-sea products; Fish products; Fish meal; Fish-egg substitutes; Preparation or treatment thereof
    • A23L17/60—Edible seaweed
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines

Definitions

  • the present invention relates to an Ecklonia cava extract for weight loss and a method for preparing the same.
  • seaweed is a good resource for the search for new functional materials.
  • Seaweed is rich in various minerals, vitamins, fiber, and protein, and is particularly rich in water-soluble polysaccharides such as alginic acid, fucan sulfate, and laminaran.
  • Ingredients contained in the algae are known to have physiological effects such as anti-diabetic, anticoagulant, anti-inflammatory and anti-tumor, but research on sea algae is insufficient and commercially available (Nishino, T. et al., A gric, Biol. Chem. , 55; 791-797, 1991)
  • Ecklonia cava is an algae and is a perennial plant of the Laminariales seaweed of the brown algae, Alariaceae. It forms a sea forest at a low-season lunch table and lives within 10 m of water. It lives on the coast of Jeju. In Japan, there is a record that food was used in ancient times. Currently, it is rarely used for food, but is used as a feed for livestock feed additives, abalone, and conch.
  • Ecklonia cava extract exhibits various physiological activities such as antibacterial and antioxidant activity, visceral fat lowering, and vasodilation (Jimenez -Escring et al., Arch.Latioam.Nutr., 49; 114-120, 1999, Marklund, S. and J. Fleurence, Trends Food Sci., 8; 22-30, 1993).
  • phlorotannin the main component of Ecklonia cava
  • Ecklonia cava is a polyphenol compound containing phloroglucinol as a basic structural unit, and physiological activities such as inhibition of thrombogenesis, antioxidant, cardiovascular protection, and antivirus have been reported (Ahn, MJ, et al., Biol. Pharm. Bull. , 27; 544-547, 2004, Kang, K., et al., Arch. Pharm. Res. , 27; 194-198, 2004).
  • Republic of Korea Patent No. 10-0762181 discloses the effect of the growth inhibitory effect of acne bacteria of Ecklonia cava extract, the Republic of Korea Patent No. 10-0771728, the activity of metalloproteinase (MMP) containing Ecklonia cava extract Cancer metastasis of the composition for inhibition, asthma and atopy prevention effect is disclosed.
  • MMP metalloproteinase
  • an object of the present invention is to provide an enzyme hydrolyzate, alcohol extract and hydrothermal extract of Ecklonia cava having an anti-obesity effect.
  • the above object of the present invention comprises the steps of preparing the Ecklonia hydrolyzate by reacting Ecklonia cava with a hydrolase to obtain Ecklonia hydrolyzate; Separately immersing Ecklonia cava in alcohol to prepare Ecklonia cava extract to obtain Ecklonia cava extract; Separately immersing Ecklonia cava in water and then extracting hot water to obtain Ecklonia cava hydrothermal extract; evaluating the effect of inhibiting adipocyte differentiation of the Ecklonia cava hydrolyzate, alcohol extract and hot water extract obtained in the above step; Eckloniatic hydrolyzate or hot water extract was ingested in mice with a high fat diet and then assessed through weight loss.
  • the present invention has an excellent effect of providing enzyme hydrolyzate, spirit extract and hydrothermal extract of Ecklonia cava having body fat and weight loss effect.
  • 1 is a graph showing the effect of reducing triglyceride content in adipocytes of four Ecklonia cava hydrolyzate prepared according to the present invention.
  • Figure 2 is a graph showing the effect of reducing the content of triglycerides in fat cells of Ecklonia cava hydrolyzate, alcohol extract and hot water extract prepared according to the present invention.
  • Figure 3 is a graph showing the cytotoxicity test results of the Ecklonia cava hydrolyzate and hot water extract prepared according to the present invention.
  • Figure 4 is a photograph of the results of analysis of the expression of fat-related protein metabolism of adipocytes treated with Ecklonia cava hydrolyzate and hot water extract prepared according to the present invention.
  • Figure 5 is a photographic result of energy metabolism-related protein expression analysis of adipocytes treated with Ecklonia cava hydrolyzate and hot water extract prepared according to the present invention.
  • Figure 6 is a graph showing the weight change of the mice respectively ingested with high-fat diet Ecklonia hydrolyzate and hot water extract prepared according to the present invention.
  • Figure 7 is a graph showing the liver weight of the mice ingested with the high-fat diet Ecklonia hydrolyzate and hot water extract prepared according to the present invention.
  • Figure 8 is a graph showing the testicular fat weight of mice ingested with the Ecklonia hydrolyzate and hot water extract prepared according to the present invention, respectively, with a high fat diet.
  • Figure 9 is a graph showing the weight of the periphery fat of the mouse ingested with the Ecklonia hydrolyzate and hot water extract prepared according to the present invention, respectively, with a high fat diet.
  • Figure 10 is a graph showing the blood glucose concentration of the mice ingested with the high-fat diet Ecklonia hydrolyzate and hot water extract prepared according to the present invention.
  • Figure 11 is a graph showing the concentration of triglycerides in the blood of mice fed with the Ecklonia hydrolyzate and hot water extract prepared according to the present invention with a high fat diet.
  • FIG. 12 is a graph showing the total cholesterol concentration in the blood of mice fed with the Ecklonia hydrolyzate and hot water extract prepared according to the present invention with a high fat diet.
  • Figure 13 is a graph showing the blood HDL-cholesterol concentration of the mouse ingested with the Ecklonia hydrolyzate and hot water extract prepared according to the present invention, respectively, with a high fat diet.
  • Figure 14 is a graph of the results of measuring the blood GOT enzyme activity of the mice ingested with the Ecklonia hydrolyzate and hot water extract prepared according to the present invention with a high fat diet.
  • Figure 15 is a graph of the results of measuring the blood GPT enzyme activity of the mice ingested with the Ecklonia hydrolyzate and hot water extract prepared according to the present invention with a high fat diet.
  • Figure 16 is a graph showing the blood insulin concentration of the mice ingested with the Ecklonia hydrolyzate and hot water extract prepared according to the present invention, respectively, with a high fat diet.
  • Figure 17 is a graph showing the blood leptin concentration of the mice ingested with the Ecklonia hydrolyzate and hot water extract prepared according to the present invention, respectively, with a high fat diet.
  • Figure 18 is a graph showing the concentration of triglycerides between the mice ingested with the Ecklonia hydrolyzate and hot water extract prepared according to the present invention with a high fat diet.
  • 19 is a graph showing the results of measuring the SOD enzyme activity between the mice ingested with the Ecklonia hydrolyzate and hot water extract prepared according to the present invention, respectively, with a high fat diet.
  • FIG. 20 is a graph showing the results of measuring the CAT enzyme activity between the mice ingested with the Ecklonia hydrolyzate and hot water extract prepared according to the present invention with a high fat diet.
  • Figure 21 is a photograph of the results of analysis of the protein expression analysis of fat metabolism between the mice ingested with the high-fat diet Ecklonia hydrolyzate and hot water extract prepared according to the present invention.
  • the enzyme for Ecklonia cava hydrolysis was used to meet the standards and standards of food additives, such as enzymes, solvents used in the manufacturing method of functional food functional ingredients in accordance with the provisions for the functional food functional raw material recognition.
  • the three enzymes used in the invention are suitable for the definition "usable microorganisms" defined in the standards and specifications of food additives.
  • Protex 6L Lapidase press L, and Lohament CL enzymes used in the present invention were purchased from Vision Biochem, Seongnam, Gyeonggi-do.
  • Protex 6L Bacillus licheniformis and Bacillus subtilis and acts rapidly on protein substrates to produce peptides
  • Rapidase press L Pectinase
  • Aspergillus niger-derived pectinase complex is a potent pectinase, cellulase, and hemicellulase complexase that increases yield in fruit vegetable juice and concentrate.
  • Rohament CL is a complex enzyme of cellulase, -glucanase and hemicellulase suitable for fruit and vegetable processing as an enzyme complex other than Trichoderma reesei-derived cellulase.
  • the Ecklonia hydrolyzate for body fat reduction is a manufacturing process including a hydrolysis reaction performed by mixing two or more enzymes selected from Protex 6L, Rapidase press L, Lohment CI enzyme or a group of these enzymes. Can be obtained.
  • the Ecklonia cava extract may be obtained by performing an extraction process including the step of extracting with an extraction solvent selected from the group consisting of water, alcohols or mixed solvents thereof.
  • the alcohols refer to ethanol, methanol, spirits, fermentation spirits and the like.
  • the amount of the extraction solvent used is not particularly limited.
  • the extraction solvent may be used in an amount of about 1 to 10 times the volume, preferably about 5 to 10 times the volume, based on 1 weight of the Ecklonia cava powder sample.
  • the Ecklonia cava extract includes all of the enzyme hydrolyzate, spirit extract and hot water extract of Ecklonia cava.
  • the present invention provides a pharmaceutical composition containing the enzyme hydrolyzate, alcohol extract and hot water extract of Ecklonia cava prepared according to the present invention as an active ingredient.
  • the enzyme hydrolyzate, ethanol extract, and hot water extract of Ecklonia cava preferably contain 0.01 to 99% by weight based on the total weight of the pharmaceutical composition.
  • the pharmaceutical composition according to the present invention may be prepared in various forms including pharmaceutically acceptable carriers, for example, oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and the like. It may be formulated in the form of sterile injectable solutions. Especially preferably, it may be formulated in an oral dosage form.
  • oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and the like. It may be formulated in the form of sterile injectable solutions. Especially preferably, it may be formulated in an oral dosage form.
  • the pharmaceutically acceptable carrier is 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, mineral oil and the like. Also included are diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, and the like.
  • Oral solid preparations include tablets, pills, powders, granules, capsules and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, sucrose or lactose. , Gelatin, and the like, and may include a lubricant such as magnesium stearate, talc, and the like.
  • Oral liquid preparations include suspensions, solvents, emulsions, syrups, and the like, and may include water, diluents such as liquid paraffin, wetting agents, sweeteners, fragrances, preservatives, and the like.
  • Parenteral preparations include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories.
  • Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and ethylol. Injectable esters such as late and the like.
  • As the base of the suppository witepsol, macrogol, tween 61, cacao butter, laurin butter, glycero gelatin and the like can be used.
  • the present invention provides a pharmaceutical composition containing the enzyme hydrolyzate, alcohol extract and hot water extract of Ecklonia cava prepared according to the present invention as an active ingredient.
  • the enzyme hydrolyzate, alcohol extract, and hot water extract of Ecklonia cauliflower should preferably contain 0.01 to 99% by weight based on the total weight of the health functional food composition.
  • the food composition of the present invention can be used as a dietary supplement.
  • health functional food means a food manufactured and processed using raw materials or ingredients having functional properties useful for the human body according to Act No. 6767 of the Health Functional Food Act, and "functionality” refers to the structure of the human body. And ingestion for the purpose of obtaining nutrients for function or for obtaining useful effects in health uses such as physiological actions.
  • the food composition of the present invention may include a conventional food additives, and the suitability as the "food additives", unless otherwise specified, in accordance with the General Regulations and General Test Methods of the Food Additives approved by the Food and Drug Administration Judge according to the standards and standards for the item.
  • Food Additive Revolution examples include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamon acid, natural additives such as color pigments, licorice extracts, crystalline cellulose, high color pigments, guar gum, And mixed preparations such as sodium L-glutamate, algae additives, preservatives and tar dyes.
  • chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamon acid
  • natural additives such as color pigments, licorice extracts, crystalline cellulose, high color pigments, guar gum
  • mixed preparations such as sodium L-glutamate, algae additives, preservatives and tar dyes.
  • the hard capsules may be prepared by filling a conventional hard capsule with a mixture of additives such as an excipient or the like, or granules or exfoliated granules thereof, and a soft capsule agent with an excipient, etc. It can be prepared by filling a mixture with an additive of a capsule base such as gelatin.
  • the soft capsule agent may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, as necessary.
  • the dietary supplement in cyclic form can be prepared by molding a mixture of excipients, binders, disintegrants, etc. in the herbal extracts in a suitable manner, and if necessary, the coating is carried out with white sugar or other suitable coating agent, or with starch, talc or a suitable substance. You may be greeted.
  • the health functional food in the form of granules may be prepared into granules in a suitable manner by mixing a mixture of an excipient, a binder, a disintegrant, and the like with the herbal extract, and may contain a flavoring agent, a copper, and the like as necessary.
  • the term definitions of the excipients, binders, disintegrants, glidants, copulation agents, flavoring agents, etc. of the present invention are described in documents known in the art and include those having the same or similar functions. Sacrament, Korean College of Pharmacy, 5 revised edition, p33-48, 1989).
  • the Ecklonia cava of the powder state used in the present invention was purchased from Taerim Co., Ltd. located in Daejeong-eup, Seogwipo-si, Jeju-do and used in the present invention.
  • the extraction efficiency according to lyophilization and spray drying showed the yield of 3-5% lower than that of freeze drying in the yield of extraction of the same amount, but in the case of spray drying, It is inevitable that the loss of oil is inevitable, and when plant-scale spray drying is performed, the yield is higher than that of small-scale spray drying. Therefore, it is superior to lyophilization in terms of yield.
  • Spray drying is considered a common method of drying. Therefore, there was no difference in yield according to the lyophilization and spray drying methods.
  • the total phenolic compound content was determined by modifying the Folin-Denis method (Swain and Hillis, 1959).
  • Ecklonia cava extract of the present invention prepared according to Preparation Examples 1 to 8 were dissolved in each extraction solvent to prepare a concentration of 0.5 mg / mL. sample 0.5 mL was added to 6.5 mL of ultrapure distilled water, and the mixture was mixed with 0.5 mL of 1N Folin-Ciocalteu ⁇ s solution and allowed to react for 3 minutes.
  • Gallic acid was measured as the standard, and the total phenolic compound content was quantified from the obtained standard curve.
  • Total phlorotannin content was determined by modifying the Folin-Ciocalteu method (Waterman and Mole, 1994). The total phlorotannin content was quantified from the standard curve obtained by the same method using Phloroglucinol as a standard.
  • Ecklonia cava extract of the present invention prepared according to Preparation Examples 1 to 8 were dissolved in each extraction solvent to prepare a concentration of 0.5 mg / mL. sample 0.1 mL was added to 1.0 mL of 1 N Folin-Ciocalteu ⁇ s solution, mixed, and allowed to stand at room temperature for 3 minutes.
  • the total polyphenol content of the eight kinds of Ecklonia cava extract according to Preparation Examples 1 to 8 was found to be 206.74 g / mL to 812.17 g / mL, Ecklonia cava extract 60% (812.17 g / mL) Extraction of organic solvents is the most effective extraction method in order to obtain polyphenols, but the yield of extracts obtained from Ecklonia cava extract is low and economic efficiency is low.
  • the extract of Ecklonia cava extract having a high content of phlorotannin is Rapidase enzyme extract Rapidase + Rohament complex enzyme extract. 60 hydrothermal extracts were found in this order.
  • 3T3-L1 pre-adipocyte cells used in the present invention were purchased from the American Cell Line Bank (American Type Culture Collection, CL-173). Cells were cultured using Dulbecco's modified Eagle's medium (DMEM) containing 10% Fetal Bovine Serum (FBS). 3T3-L1 pre-adipocytes are known to be suitable cell models for adipocyte-related experiments by adding differentiation-inducing factors, since they proliferate and induce differentiation when cells mature.
  • DMEM Dulbecco's modified Eagle's medium
  • FBS Fetal Bovine Serum
  • Serum-free medium SFM was treated for 24 hours, followed by 250, 500 g of each of the Ecklonia cava extract samples prepared according to Preparation Examples 1 to 4 above. Treatment was performed for 24 hours at / mL concentration. After 24 hours of sample treatment, each cell treated with the sample was washed twice with PBS, added with ice-cold PBS 500, and sonicated for 30 seconds to allow the contents in the cell membrane to be eluted. TG content of the cell lysate obtained by the ultrasonic grinding was analyzed using TG measuring reagent of Asan Pharmaceutical.
  • TG assay reagent 200 was added to the cell lysate 10, mixed, and then reacted at 37 for 10 minutes, and then measured at 550 nm absorbance using a multi-well plate reader.
  • the cell treatment concentration was determined as 250g / mL concentration because it showed a similar effect to the concentration of 500g / mL even at the concentration of 250g / mL.
  • Samples used for measuring cell viability are effective in weight loss, and the conventionally reported garcinia extract and phloroglucinol (ALDRICH 79330), a phloroglucin basic structure, were set as positive controls, respectively, and Preparation Example 4 (R + R enzyme extract). ), Cell viability assay of the sample prepared according to Preparation Example 8 (60 hot water extract).
  • 3T3-L1 cells were seeded at a concentration of 2 4 cells / well in a 96-well plate and incubated for 20 hours. Each cell was treated with SFM for 4 hours, followed by Garcinia extract (G), Phloroglucinol (P), Preparation Example 4 (Rapidase + Rohament; R + R), and Preparation Example 8 (60 hydrothermal extract; 60 H). The cells were treated with cells at concentrations of 12.5 50 and 200 g / mL and then incubated for 24 and 48 hours.
  • the cell survival rate decreased with the concentration of the positive control Phloroglucinol, but showed a tendency to decrease above the negative control (CON) level, did not show a decrease in the cell survival rate according to the sample treatment. Therefore, in the subsequent experiment, the experiment was conducted based on 24 hours.
  • Differentiation was induced for 6 days by seeding 3T3-L1 cells in a 6-well plate and processing 80% confluent. After induction of differentiation, the culture medium of each cell is removed and replaced with Serum-free medium (SFM) to incubate cells in serum-free medium for 24 hours. 50 g / mL Garcinia extract (G), Rapidase + Rohament (R + R; Preparation 4), 60 hydrothermal extracts (60 H; Preparation 8) were added to the cells in serum-free medium. Treatment was performed for 24 hours at a concentration of / mL.
  • SFM Serum-free medium
  • each of the recovered cell lysates was subjected to protein quantification, and then the same amount of protein was electrophoresed on 815% gel to transfer to PVDF membrane.
  • the PVDF membrane to which the protein lysate of each cell was transferred was blocked with 1% BSA-TBS-T for 1 hour, and then the primary antibody (1: 1000, 1%) of each protein to be analyzed on the blocked PVDF membrane.
  • BSA-TBS-T was reacted at 10 to 16 hours and then washed with TBS-T.
  • Secondary antibodies (1: 10000) were attached to the washed PVDF membrane, respectively, and developed with KODAK X-ray and X-OMAT films with Chemiluminescent sensitive HRP. The results are shown in FIGS. 4 to 5.
  • the protein analyzed in this example was Acrp30, which is used as an indicator of metabolic syndrome, Ob (leptin), fat involved in maintaining energy homeostasis and weight control.
  • Ob lactin
  • PPAR and C / EBP involved in cell differentiation and fatty acid metabolism regulation, aP2 a marker for adipocyte differentiation, SREBP-1, which regulates the expression of genes related to cholesterol, fatty acid and triglyceride metabolism, FAS involved in fatty acid production mechanism, LPL and pACC-proteins.
  • SREBP-1 a marker for adipocyte differentiation
  • 3T3-L1 adipocytes treated with Ecklonia cava extract prepared according to Preparation Examples 4 and 8 of the present invention increased the expression of Ob protein in the MDI treatment group, and R + R (Preparation Example 4) treatment group and Expression was reduced in a concentration dependent manner in the 60 H (Preparation Example 8) group.
  • ACC is a protein whose activity is regulated according to phosphorylation (inactivation) and dephosphorylation (activation) of serin residues, and the concentration of ACC- when Ecklonia cava extracts (Preparation Example 4 and Preparation Example 8) were treated in adipocytes by concentration. Phosphorylation levels decreased concentration dependently.
  • the Ecklonia cava extract prepared according to the present invention is considered to have an excellent effect of inhibiting adipocyte differentiation and adipogenesis.
  • the protein analyzed in this experiment is activated by growth hormones such as SOCS-3 and EGF that regulate cytokine signaling.
  • growth hormones such as SOCS-3 and EGF that regulate cytokine signaling.
  • Stat3 and phosphorylated Stat3, UCP2 involved in thermoregulation and sugar metabolism GLUT4 protein is a sugar transport protein. Expression analysis results of the proteins are shown in FIG.
  • Anti-obesity activity assay of Ecklonia cava extract prepared according to Preparation Example 4 and Preparation Example 8 of the present invention was carried out using an animal in which C57BL / 6 mouse was bred through a high-fat diet for 10 weeks.
  • each sample was used for animals as shown in Table 5 below. It was added to the feed by concentration to the animals.
  • the garcinia extract was used as a positive control to compare the effects of the Ecklonia cava extract of the present invention.
  • the composition of the experimental diet is shown in Table 4 below.
  • the Normal Diet was formulated according to the AIN-76 semipurified diet (MP 0290545220) (corn oil, 5% wt / wt) and the high fat diet (HFD) was lard and corn oil ( 17: 5) was used as a lipid source, and the dietary fat content was adjusted to 20% (37% of total calories). According to other previous studies, when dietary fat is added at 20% wt / wt level, 40% of total calories are supplied to fat source, which is about 20% compared to the dietary patterns of Koreans fed from lipids. The experiment was conducted as judged to be suitable for the obesity model.
  • the Ecklonia cava sample was prepared to be 5mg, 25mg, 150mg / 20kcal / day.
  • mice at the start of animal experiment was 20.8 0.44g
  • the change in body weight after 10 weeks of breeding was normal diet (ND) 32.0, high fat diet (HD) 36.2, garcinia extract diet (GAR 25).
  • ND normal diet
  • HD high fat diet
  • GAR 25 garcinia extract diet
  • E. cava 60H Ecklonia cava 60H
  • E. cava 60H Ecklonia cava R + R Enzyme-treated diet
  • E. cava R + R Enzyme-treated diet
  • weight loss was significantly decreased in 150 mg of Ecklonia 60 hot water extract (Preparation Example 8) and 150 mg of Ecklonia cava R + R enzyme treatment (Preparation Example 4) compared to the normal diet group.
  • mice were fasted for one day, and liver, epididymal fat, and fat from the kidneys were extracted under anesthesia with diethyl ether.
  • the weight of liver tissue showed the same tendency as the result of the previous weight.
  • the weight of liver tissue was significantly decreased in the 150 mg group of Ecklonia cava 60 hot water extract and the 150 mg Ecklonia cava R + R enzyme treatment group compared with the normal diet group.
  • the weight of epididymal fat was reduced to the level of weight similar to that of the normal diet group in the 150 mg diet group of the Ecklonia cava hot-water extract and the R + R enzyme-treated 150 mg diet group.
  • Figure 9 in the fat weight around the kidney did not show a significant decrease by the treatment of the Ecklonia cava extract of the present invention.
  • mice were fasted for one day, and then anesthetized with diethyl ether to collect blood, and the blood was centrifuged at 2,500 rpm for 20 minutes to separate serum, and sugar, Cholesterol, HDL-cholesterol and tiglyceride in serum. And leptin concentrations were measured.
  • the serum obtained from blood collection on the final autopsy day was measured using a blood glucose measurement kit (Asan Pharmaceutical Co., AM 201-K) to measure the concentration of GLUCOSE in mice. After making a determination line through, 20ul of frozen serum was taken and measured at 500nm with a spectrophotometer.
  • the blood sugar level was significantly increased in the high-fat diet group (HD) compared to the normal diet group (ND), and the garcinia group (GAR 25) used as a positive control group, and the Eckonia enzyme treatment group (R + R 150), and EEG 150 mg (60H 150) treated group significantly decreased blood glucose levels.
  • HD high-fat diet group
  • ND normal diet group
  • GAR 25 garcinia group
  • R + R 150 Eckonia enzyme treatment group
  • EEG 150 mg (60H 150) treated group significantly decreased blood glucose levels.
  • the blood triglyceride content was significantly increased in the high fat diet group (HD) as compared to the normal diet group (ND), and the garcinia group (GAR 25) used as a positive control group, Ecklonia hot-water extract In the 150 mg (60H 150; Preparation Example 8) group and Eckloniasis enzyme treatment group (R + R 150; Preparation Example 4) group, the triglyceride content was reduced in a concentration-dependent manner.
  • HD high fat diet group
  • ND normal diet group
  • GAR 25 garcinia group
  • R + R 150 Eckloniasis enzyme treatment group
  • the total cholesterol content was significantly increased in the high fat diet group (HD) compared to the normal diet group (ND), and the total cholesterol in the Ecklonia cava hot-water extract treatment group (Preparation Example 8) of the present invention.
  • the content was found to decrease. .
  • the Ecklonia cava extract 150mg of the present invention (Preparation Example 8) and the enzyme treatment 150mg dietary group of the present invention (Preparation Example 4) increased as shown in Figure 13, the present invention Ecklonia cava extract (Preparation Example) 4, Preparation Example 8)
  • the group treated with 25mg respectively HDL-cholesterol was increased compared to the total cholesterol, so it is believed that the Ecklonia cava extract of the present invention will have a body fat reduction effect.
  • GOT / GPT was measured using Ritman-Frankel method for kit (Asan Pharmaceutical Co., AM 101-K), and 200ul of serum stored in frozen serum was prepared by using a standard solution based on 1 ml of substrate solution. GOT was reacted for 60 minutes and GPT for 30 minutes in 37 incubators. After mixing with 1 ml of color solution, the mixture was allowed to stand at room temperature for 20 minutes, and 10 ml of 0.4N sodium hydroxide solution was mixed well for 10 minutes at room temperature, and distilled water was measured as a control at 505 nm on a spectrophotometer.
  • GOT / GPT is an enzyme that is present in blood at high levels when the liver is damaged. An increase in GOT / GPT activity in serum indicates liver damage.
  • Insulin concentration was measured by using a mouse insulin ELISA kit (catalog NO 80-INSMS-Eol) was used to quantify the serum insulin concentration at the time of autopsy. After the determination line was prepared using the standard solution, 10ul of the frozen stored serum was dispensed by 75ul of each working strength conjugate, and then left at room temperature for 2 hours (700-900 rpm). Thereafter, 100 ⁇ l of TMB substrate was dispensed and shaken at room temperature for 15 minutes. A 100ul stop solution was taken to measure distilled water at 450 nm as a control in a spectrophotometer.
  • Insulin plays a role in regulating blood sugar and is known to increase the risk of obesity in adipose tissue.
  • the effect of high-fat diet on serum insulin concentration was analyzed.
  • the insulin content was significantly increased in the high fat diet group (HD) compared to the normal diet group (ND), as shown in Figure 16, the blood content of insulin in all the Ecklonia cava treatment group of the present invention Significantly decreased.
  • the Ecklonia cava extract of the present invention was confirmed to have an excellent effect of lowering the increased insulin content due to high fat diet.
  • Serum leptin concentrations were quantified using leptin ELISA kit (catalog NO ADI-900-019A) using serum isolated during autopsy to determine the leptin content used by Ecklonia cava extract as a marker of differentiation of adipocytes. After making the determination line using the standard solution to 100ul assay buffer, 100ul of frozen serum was taken and allowed to shake for 1 hour at room temperature.
  • washing with PBS solution was repeated three times, followed by dispensing blue conjugates at 100 ul for 30 minutes.
  • 100ul of TMB substrate solution was dispensed and reacted for 30 minutes at room temperature.
  • 100ul of stop solution was taken and distilled water was measured as a control in a spectrophotometer 450nm.
  • Leptin is produced and secreted mainly from fat cells, and its amount is known to be proportional to body fat mass.
  • liver tissue was extracted, washed with physiological saline solution, water was removed with filter paper, frozen with liquid nitrogen and stored at 70 to use as liver tissue sample. It was. The obtained liver tissue was cut and put into PBS buffer, homogenized, and centrifuged at 10,000, 4, and 15 min, and the supernatant was used.
  • the triglycerides of liver tissue were measured using kit (Asan Pharmaceutical Co., AM 157S-K), and after making the determination line using the standard solution based on 3 ml of enzyme solution, take 20ul of homogenized liver tissue stored in frozen form 37 After standing in the incubator for 10 minutes, it was measured at 550 nm spectrophotometer.
  • triglyceride level of liver was significantly increased in the high fat diet group (HD) compared to the normal diet group (ND), and the present invention Ecklonia cava fermentation extract (Preparation Example 8) and hydrolyzate ( Preparation Example 4
  • the triglyceride content was significantly decreased in the diet group.
  • SOD is distributed in almost all tissues of animals, and is known to exist most in liver tissue. It is an enzyme that plays an important role in protecting cells and aerobic organisms from oxidative stress, and its mechanism of action is associated with the redox of metals in the enzymes to transfer electrons from superoxide radicals to remove toxic superoxide radicals. It is known.
  • SOD activity analysis in the liver tissue was used kit (cayman, 706002), the liver tissue of the experimental animal performed in accordance with Example 4 were extracted respectively SOD extraction buffer (20mM HEPES buffer, pH7.2, containing 1mM EGTA , 210mM mannitol, and 70mM sucrose) , The supernatant obtained by centrifugation for 4 to 5 minutes was used as the enzyme source. 10ul of sample and SOD standard were dispensed into the 96-well plate, and 200ul of diluted radical detector was added, followed by adding 20ul of xantine oxidase. After shaking for 20 minutes at room temperature, Spectrophotometer at 450 nm SOD activity in liver tissue was measured.
  • SOD antioxidants are known to play a role in scavenging free radicals generated during metabolism by converting hydrogen peroxide into harmless water and oxygen by GPx, which converts superoxide radicals to hydrogen peroxide and then detoxifies them. .
  • CAT activity analysis in liver tissue was performed using kit (cayman, 707002). Liver tissues of the experimental animals carried out according to Example 4 were extracted, respectively, for CAT extraction buffer (50 mM potassium phosphate, pH 7.0, containing 1 mM EDTA). ) It was homogenized to, and centrifuged for 15 minutes at 10,000, 4, and then the supernatant was collected and used. In the measurement method, 20ul of the supernatant, Formaldehyde standard, and positive control were each dispensed, and 100ul of 1X assay buffer and 30ul of MeOH were added, and 20ul of hydrogen peroxide was added thereto and left at room temperature for 20 minutes.
  • CAT extraction buffer 50 mM potassium phosphate, pH 7.0, containing 1 mM EDTA.
  • CAT enzyme activity in liver tissue was reduced in the high-fat diet group compared to the normal diet group, in the Ecklonia cava enzyme extract treatment group prepared according to Preparation Examples 4 and 8 in the present invention CAT enzyme activity was restored to normal levels.
  • the Ecklonia cava extract of the present invention is believed to restore liver function by increasing the reduced CAT enzyme activity in a high fat diet.
  • Liver tissues of the experimental animals performed according to Example 4 were extracted, and the protein expression levels of the obesity related factors were analyzed by western blotting.
  • ACC-1 and FAS genes which are known to be involved in fat synthesis, were reduced in the Ecklonia cava diet group compared to the high fat diet group, but PPAR-, which is known to be involved in lipolysis,
  • the CD-36 gene showed a tendency to increase in the ECO diet compared to the high-fat diet, which was increased in the enzyme treatment group.
  • OB-receptor increased with obesity tended to decrease in EAC group compared to high fat diet group.Acrp30 (adiponectin) activates AMP Kinase in liver and muscle to inhibit ACC-1 expression and reduce fatty acid oxidation. It is known to play a role in promoting. Contrary to the results of the above ACC-1, it was found to increase in the Ecklonia cava group, thereby promoting fatty acid oxidation.
  • Ecklonia cava hot water extract and enzyme-treated extract to a high-fat diet regulates the expression level of fat metabolism-related protein, lowers insulin content to regulate blood sugar, and also affects cholesterol metabolism such as triglycerides, obesity It can be used as an anti-obesity material because it has a positive effect on improving lipid metabolism abnormalities.
  • the present invention has an excellent effect of providing enzyme hydrolyzate, fermented alcohol extract and hot water extract of Ecklonia cava having a weight loss effect and anti-obesity effect, and thus is a very useful invention for the health functional food industry and the biopharmaceutical industry.

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Abstract

The present invention has an excellent effect of providing an enzyme hydrolysate, a fermented alcohol extract and a hot water extract of Ecklonia cava which has weight loss and anti-obesity effects.

Description

체중 감소용 감태 추출물 및 이의 제조방법Ecklonia cava extract for weight loss and preparation method thereof
본 발명은 체중 감소용 감태 추출물 및 이의 제조방법에 관한 것이다.The present invention relates to an Ecklonia cava extract for weight loss and a method for preparing the same.
급격한 생활환경의 변화와 서구화된 식생활로 인하여 당뇨병 및 비만 등과 같은 대사성 질환이 증가 되고, 이를 예방하기 위해 안전성이 입증된 천연물에 대한 연구가 이루어지고 있다. 그러나 이러한 연구는 육상 식물을 대상으로 하는 것이 대부분이며, 이미 새로운 기능성 소재개발에 한계성이 문제점으로 지적되고 있다(Hye-youn Kim et al.,Journal of Life Science, 22(6);751-759, 2012).Due to the rapid change in living environment and westernized diet, metabolic diseases such as diabetes and obesity are increasing, and research on natural products that have been proven safe to prevent them is being conducted. However, most of these studies have been conducted on land plants, and limitations in the development of new functional materials have already been pointed out as problems (Hye-youn Kim et al., Journal of Life Science , 22 (6); 751-759, 2012).
따라서, 해조류는 새로운 기능성소재 탐색을 위한 좋은 자원이다. 해조류에는 각종 미네랄과 비타민 및 섬유소, 단백질이 풍부하게 함유되어 있으며, 특히 알긴산, 퓨칸설페이트, 라미나란 등 수용성 다당류가 풍부하다. 상기 해조류에 함유된 성분들은 항당뇨, 항응고, 항염증, 항종양 등의 생리 효능이 알려져 있으나 해조류에 대한 연구가 미흡하여, 상업적으로 이용이 미흡한 실정이다(Nishino, T. et al., Agric, Biol. Chem., 55;791-797, 1991) Thus, seaweed is a good resource for the search for new functional materials. Seaweed is rich in various minerals, vitamins, fiber, and protein, and is particularly rich in water-soluble polysaccharides such as alginic acid, fucan sulfate, and laminaran. Ingredients contained in the algae are known to have physiological effects such as anti-diabetic, anticoagulant, anti-inflammatory and anti-tumor, but research on sea algae is insufficient and commercially available (Nishino, T. et al., A gric, Biol. Chem. , 55; 791-797, 1991)
감태(Ecklonia cava)는 해조류로 갈조식물문의 갈조강 다시마목(Laminariales) 미역과(Alariaceae)의 다년생 대형식물로 저조선하의 점심대에서 바다숲을 형성하며 수심 10 m 이내에서 서식하여 주로 일본과 우리나라 제주 연안에 서식한다. 일본에서는 고대에 식용으로 사용하였다는 기록이 있으며, 현재는 식용으로는 거의 사용되지 않고 가축 사료첨가제와 전복, 소라 양식을 위한 먹이로 사용되고 있다.Ecklonia cava is an algae and is a perennial plant of the Laminariales seaweed of the brown algae, Alariaceae. It forms a sea forest at a low-season lunch table and lives within 10 m of water. It lives on the coast of Jeju. In Japan, there is a record that food was used in ancient times. Currently, it is rarely used for food, but is used as a feed for livestock feed additives, abalone, and conch.
감태에 강력한 세포보호 및 활성화 능력을 가진 폴리페놀 성분이 함유되어 있으며, 감태 추출물이 항균 및 항산화 작용, 내장지방의 저하 작용, 혈관 확장 작용 등 다양한 생리 활성을 나타낸다는 연구 결과가 보고되고 있다(Jimenez-Escring et al., Arch. Latioam. Nutr., 49;114-120, 1999, Marklund, S. and J. Fleurence, Trends Food Sci., 8;22-30, 1993).Polyphenols, which have strong cytoprotective and activating abilities, are contained in Ecklonia cava, and studies have shown that Ecklonia cava extract exhibits various physiological activities such as antibacterial and antioxidant activity, visceral fat lowering, and vasodilation (Jimenez -Escring et al., Arch.Latioam.Nutr., 49; 114-120, 1999, Marklund, S. and J. Fleurence, Trends Food Sci., 8; 22-30, 1993).
특히, 감태의 주요 성분인 phlorotannin은 phloroglucinol을 기본 구성단위로 하는 폴리페놀화합물로서 혈전생성 저해, 항산화, 심혈관 보호, 항바이러스 등의 생리활성이 보고되어 있다(Ahn, M. J., et al., Biol. Pharm. Bull., 27;544-547, 2004, Kang, K., et al., Arch. Pharm. Res., 27;194-198, 2004). In particular, phlorotannin, the main component of Ecklonia cava, is a polyphenol compound containing phloroglucinol as a basic structural unit, and physiological activities such as inhibition of thrombogenesis, antioxidant, cardiovascular protection, and antivirus have been reported (Ahn, MJ, et al., Biol. Pharm. Bull. , 27; 544-547, 2004, Kang, K., et al., Arch. Pharm. Res. , 27; 194-198, 2004).
감태의 생리활성과 관련된 종래의 기술로서는 대한민국 특허출원 제10-2008-0118374호에 천식완화 효과가 있는 감태추출물을 포함하는 조성물이 개시되어 있고, 대한민국 특허출원 제10-2011-0034398호에 감태 추출물을 함유하는 알레르기성 피부질환 증상 개선용 조성물이 개시되어 있다.Conventional techniques related to the physiological activity of Ecklonia cava are disclosed in Korean Patent Application No. 10-2008-0118374, which contains Ecklonia cava extract with asthma relief effect, and Ecklonia cava extract in Korean Patent Application No. 10-2011-0034398. Disclosed is a composition for improving allergic skin disease symptoms containing.
또, 대한민국 등록특허 제10-0762181호에 감태 추출물의 여드름균의 성장 저해능에 대한 효능이 개시되어 있으며, 대한민국 등록특허 제10-0771728호에는 감태추출물을 포함하는 금속단백질분해효소(MMP)의 활성 억제용 조성물의 암전이, 천식 및 아토피 예방 효과가 개시되어 있다.In addition, the Republic of Korea Patent No. 10-0762181 discloses the effect of the growth inhibitory effect of acne bacteria of Ecklonia cava extract, the Republic of Korea Patent No. 10-0771728, the activity of metalloproteinase (MMP) containing Ecklonia cava extract Cancer metastasis of the composition for inhibition, asthma and atopy prevention effect is disclosed.
그러나, 감태의 효소 가수분해물, 주정 추출물 및 열수 추출물의 항비만효능에 대해서는 보고된 바 없다.However, the anti-obesity effect of enzyme hydrolyzate, alcohol extract and hydrothermal extract of Ecklonia cava has not been reported.
따라서, 본 발명의 목적은 항비만 효과를 갖는 감태의 효소 가수분해물, 주정 추출물 및 열수 추출물을 제공하는 데 있다.Accordingly, an object of the present invention is to provide an enzyme hydrolyzate, alcohol extract and hydrothermal extract of Ecklonia cava having an anti-obesity effect.
본 발명의 상기 목적은, 감태를 가수분해 효소와 반응시켜 감태 가수분해물을 제조하여 감태 가수분해물을 수득하는 단계와; 이와 별도로 감태를 주정에 침지하여 감태 주정 추출물을 제조하여 감태 주정추출물을 수득하는 단계와; 이와 별도로 감태를 물에 침지한 다음 열수 추출하여 감태 열수 추출물을 수득하는 단계와;상기 단계에서 수득한 감태 가수분해물, 주정추출물 및 열수 추출물의 지방세포분화 억제효과를 평가하는 단계와; 감태 가수분해물 또는 열수추출물을 고지방식이와 함께 마우스에 섭취시킨 다음 체중감소효과를 평가하는 단계를 통하여 달성하였다.The above object of the present invention comprises the steps of preparing the Ecklonia hydrolyzate by reacting Ecklonia cava with a hydrolase to obtain Ecklonia hydrolyzate; Separately immersing Ecklonia cava in alcohol to prepare Ecklonia cava extract to obtain Ecklonia cava extract; Separately immersing Ecklonia cava in water and then extracting hot water to obtain Ecklonia cava hydrothermal extract; evaluating the effect of inhibiting adipocyte differentiation of the Ecklonia cava hydrolyzate, alcohol extract and hot water extract obtained in the above step; Eckloniatic hydrolyzate or hot water extract was ingested in mice with a high fat diet and then assessed through weight loss.
본 발명은 체지방 및 체중 감소 효과를 갖는 감태의 효소 가수분해물, 주정 추출물 및 열수 추출물을 제공하는 뛰어난 효과가 있다.The present invention has an excellent effect of providing enzyme hydrolyzate, spirit extract and hydrothermal extract of Ecklonia cava having body fat and weight loss effect.
도 1은 본 발명에 따라 제조된 감태 가수분해물 4종의 지방세포내 중성지방 함량 감소효과를 나타낸 그래프이다.1 is a graph showing the effect of reducing triglyceride content in adipocytes of four Ecklonia cava hydrolyzate prepared according to the present invention.
도 2는 본 발명에 따라 제조된 감태 가수분해물, 주정추출물 및 열수추출물의 지방세포내 중성지방 함량 감소효과를 나타낸 그래프이다.Figure 2 is a graph showing the effect of reducing the content of triglycerides in fat cells of Ecklonia cava hydrolyzate, alcohol extract and hot water extract prepared according to the present invention.
도 3은 본 발명에 따라 제조된 감태 가수분해물 및 열수추출물의 세포독성실험 결과를 나타낸 그래프이다.Figure 3 is a graph showing the cytotoxicity test results of the Ecklonia cava hydrolyzate and hot water extract prepared according to the present invention.
도 4는 본 발명에 따라 제조된 감태 가수분해물 및 열수추출물을 각각 처리한 지방세포의 지방대사관련 단백질 발현분석 결과 사진도이다.Figure 4 is a photograph of the results of analysis of the expression of fat-related protein metabolism of adipocytes treated with Ecklonia cava hydrolyzate and hot water extract prepared according to the present invention.
도 5는 본 발명에 따라 제조된 감태 가수분해물 및 열수추출물을 각각 처리한 지방세포의 에너지대사관련 단백질 발현분석 결과 사진도이다.Figure 5 is a photographic result of energy metabolism-related protein expression analysis of adipocytes treated with Ecklonia cava hydrolyzate and hot water extract prepared according to the present invention.
도 6은 본 발명에 따라 제조된 감태 가수분해물 및 열수추출물을 고지방식이와 함께 각각 섭취한 마우스의 체중변화를 나타낸 그래프이다.Figure 6 is a graph showing the weight change of the mice respectively ingested with high-fat diet Ecklonia hydrolyzate and hot water extract prepared according to the present invention.
도 7은 본 발명에 따라 제조된 감태 가수분해물 및 열수추출물을 고지방식이와 함께 각각 섭취한 마우스의 간 무게를 나타낸 그래프이다.Figure 7 is a graph showing the liver weight of the mice ingested with the high-fat diet Ecklonia hydrolyzate and hot water extract prepared according to the present invention.
도 8은 본 발명에 따라 제조된 감태 가수분해물 및 열수추출물을 고지방식이와 함께 각각 섭취한 마우스의 고환지방무게를 나타낸 그래프이다.Figure 8 is a graph showing the testicular fat weight of mice ingested with the Ecklonia hydrolyzate and hot water extract prepared according to the present invention, respectively, with a high fat diet.
도 9는 본 발명에 따라 제조된 감태 가수분해물 및 열수추출물을 고지방식이와 함께 각각 섭취한 마우스의 신장주변 지방의 무게를 나타낸 그래프이다.Figure 9 is a graph showing the weight of the periphery fat of the mouse ingested with the Ecklonia hydrolyzate and hot water extract prepared according to the present invention, respectively, with a high fat diet.
도 10은 본 발명에 따라 제조된 감태 가수분해물 및 열수추출물을 고지방식이와 함께 각각 섭취한 마우스의 혈중 당농도를 나타낸 그래프이다.Figure 10 is a graph showing the blood glucose concentration of the mice ingested with the high-fat diet Ecklonia hydrolyzate and hot water extract prepared according to the present invention.
도 11은 본 발명에 따라 제조된 감태 가수분해물 및 열수추출물을 고지방식이와 함께 각각 섭취한 마우스의 혈중 중성지방 농도를 나타낸 그래프이다.Figure 11 is a graph showing the concentration of triglycerides in the blood of mice fed with the Ecklonia hydrolyzate and hot water extract prepared according to the present invention with a high fat diet.
도 12는 본 발명에 따라 제조된 감태 가수분해물 및 열수추출물을 고지방식이와 함께 각각 섭취한 마우스의 혈중 총콜레스테롤 농도를 나타낸 그래프이다.12 is a graph showing the total cholesterol concentration in the blood of mice fed with the Ecklonia hydrolyzate and hot water extract prepared according to the present invention with a high fat diet.
도 13은 본 발명에 따라 제조된 감태 가수분해물 및 열수추출물을 고지방식이와 함께 각각 섭취한 마우스의 혈중 HDL-콜레스테롤 농도를 나타낸 그래프이다.Figure 13 is a graph showing the blood HDL-cholesterol concentration of the mouse ingested with the Ecklonia hydrolyzate and hot water extract prepared according to the present invention, respectively, with a high fat diet.
도 14는 본 발명에 따라 제조된 감태 가수분해물 및 열수추출물을 고지방식이와 함께 각각 섭취한 마우스의 혈중 GOT 효소활성을 측정한 결과 그래프이다.Figure 14 is a graph of the results of measuring the blood GOT enzyme activity of the mice ingested with the Ecklonia hydrolyzate and hot water extract prepared according to the present invention with a high fat diet.
도 15는 본 발명에 따라 제조된 감태 가수분해물 및 열수추출물을 고지방식이와 함께 각각 섭취한 마우스의 혈중 GPT 효소활성을 측정한 결과 그래프이다.Figure 15 is a graph of the results of measuring the blood GPT enzyme activity of the mice ingested with the Ecklonia hydrolyzate and hot water extract prepared according to the present invention with a high fat diet.
도 16은 본 발명에 따라 제조된 감태 가수분해물 및 열수추출물을 고지방식이와 함께 각각 섭취한 마우스의 혈중 인슐린 농도를 나타낸 그래프이다.Figure 16 is a graph showing the blood insulin concentration of the mice ingested with the Ecklonia hydrolyzate and hot water extract prepared according to the present invention, respectively, with a high fat diet.
도 17은 본 발명에 따라 제조된 감태 가수분해물 및 열수추출물을 고지방식이와 함께 각각 섭취한 마우스의 혈중 렙틴 농도를 나타낸 그래프이다.Figure 17 is a graph showing the blood leptin concentration of the mice ingested with the Ecklonia hydrolyzate and hot water extract prepared according to the present invention, respectively, with a high fat diet.
도 18은 본 발명에 따라 제조된 감태 가수분해물 및 열수추출물을 고지방식이와 함께 각각 섭취한 마우스 간의 중성지방 농도를 나타낸 그래프이다.Figure 18 is a graph showing the concentration of triglycerides between the mice ingested with the Ecklonia hydrolyzate and hot water extract prepared according to the present invention with a high fat diet.
도 19는 본 발명에 따라 제조된 감태 가수분해물 및 열수추출물을 고지방식이와 함께 각각 섭취한 마우스 간의 SOD 효소활성을 측정한 결과 그래프이다.19 is a graph showing the results of measuring the SOD enzyme activity between the mice ingested with the Ecklonia hydrolyzate and hot water extract prepared according to the present invention, respectively, with a high fat diet.
도 20은 본 발명에 따라 제조된 감태 가수분해물 및 열수추출물을 고지방식이와 함께 각각 섭취한 마우스 간의 CAT 효소활성을 측정한 결과 그래프이다.20 is a graph showing the results of measuring the CAT enzyme activity between the mice ingested with the Ecklonia hydrolyzate and hot water extract prepared according to the present invention with a high fat diet.
도 21은 본 발명에 따라 제조된 감태 가수분해물 및 열수추출물을 고지방식이와 함께 각각 섭취한 마우스 간의 지방대사관련 단백질 발현분석 결과 사진도이다.Figure 21 is a photograph of the results of analysis of the protein expression analysis of fat metabolism between the mice ingested with the high-fat diet Ecklonia hydrolyzate and hot water extract prepared according to the present invention.
본 발명에 있어서, 감태 가수분해용 효소는건강기능식품 기능성 원료 인정에 관한 규정에 따라 건강기능식품 기능성 원료의 제조방법에 사용되는 효소, 용매 등식품첨가물의 기준 및 규격에 적합한 것을 사용하였으며, 본 발명에 사용한 3종의 효소는 식품첨가물의 기준 및 규격에서 정한 정의 "사용 가능한 미생물"에 적합하다. In the present invention, the enzyme for Ecklonia cava hydrolysis was used to meet the standards and standards of food additives, such as enzymes, solvents used in the manufacturing method of functional food functional ingredients in accordance with the provisions for the functional food functional raw material recognition. The three enzymes used in the invention are suitable for the definition "usable microorganisms" defined in the standards and specifications of food additives.
본 발명에 사용한 프로텍스 6엘, 라피다제 프레스 엘, 로하멘트 씨엘 효소 3종은 경기도 성남시 소재 비전바이오켐에서 구매하였다. 프로텍스 6엘(Protex 6L, 세균성 프로테아제)은 Bacillus licheniformis 및 Bacillus subtilis 유래 세균성 endo-type 프로테아제이고 단백질 기질에 빠른 속도로 작용하여 펩타이드 생성하고, 라피다제 프레스 엘(Rapidase press L, 펙티나아제)는 Aspergillus niger 유래 펙티나아제 복합물로서 과채류 착즙 및 농축 시 수율을 증대시키는 강력한 펙티나아제, 셀룰라아제, 헤미셀룰라아제 복합효소이다. 또, 로하멘트 씨엘(Rohament CL, 셀룰라아제)은 Trichoderma reesei 유래 셀룰라아제 외 효소 복합물로서 과채류 가공에 적합한 cellulase, -glucanase와 hemicellulase의 복합효소이다.Protex 6L, Lapidase press L, and Lohament CL enzymes used in the present invention were purchased from Vision Biochem, Seongnam, Gyeonggi-do. Protex 6L (Bacterial Protease) is a bacterial endo-type protease derived from Bacillus licheniformis and Bacillus subtilis and acts rapidly on protein substrates to produce peptides, and Rapidase press L (Pectinase) Aspergillus niger-derived pectinase complex is a potent pectinase, cellulase, and hemicellulase complexase that increases yield in fruit vegetable juice and concentrate. Rohament CL (Cellulase) is a complex enzyme of cellulase, -glucanase and hemicellulase suitable for fruit and vegetable processing as an enzyme complex other than Trichoderma reesei-derived cellulase.
상기 기재한 3종의 가수분해 효소의 반응 조건은 하기 표 1에 나타냈다.The reaction conditions of the above-described three kinds of hydrolase are shown in Table 1 below.
표 1 본 발명에 사용한 가수분해 효소의 반응 조건
Enzyme Optimum conditions Enzyme characteristics
pH Temp.()
Protex 6L 7.0 60 세균성 프로테아제, 실활(90, 15분 유지)
Rapidase press L 4.5 50 펙티나아제
Rohament CL 4.5 50 셀룰라아제
Table 1 Reaction conditions of the hydrolase used in the present invention
Enzyme Optimum conditions Enzyme characteristics
pH Temp. ()
Protex 6L 7.0 60 Bacterial Protease, Inactivation (90 minutes, 15 minutes)
Rapidase press L 4.5 50 Pectinase
Rohament CL 4.5 50 Cellulase
본 발명에 따르면, 체지방 감소용 감태 가수분해물은 프로텍스 6엘, 라피다제 프레스 엘, 로하멘트 씨엘 효소 또는 이들 효소군 중 선택되는 2종 이상 효소를 혼합하여 수행한 가수분해반응을 포함하는 제조과정으로 얻어질 수 있다. According to the present invention, the Ecklonia hydrolyzate for body fat reduction is a manufacturing process including a hydrolysis reaction performed by mixing two or more enzymes selected from Protex 6L, Rapidase press L, Lohment CI enzyme or a group of these enzymes. Can be obtained.
본 발명에 따르면, 감태 추출물은 물, 알코올류 또는 이들의 혼합용매로 이루어진 군으로부터 선택된 추출용매로 추출하는 단계를 포함하는 추출공정을 수행하여 얻어질 수 있다. 상기 알코올류란 에탄올, 메탄올, 주정, 발효주정 등을 말한다.According to the present invention, the Ecklonia cava extract may be obtained by performing an extraction process including the step of extracting with an extraction solvent selected from the group consisting of water, alcohols or mixed solvents thereof. The alcohols refer to ethanol, methanol, spirits, fermentation spirits and the like.
상기 추출용매의 사용량은 크게 제한되는 것은 아니며, 예를 들어 감태 분말 시료 1 중량에 대하여 추출용매를 약 1 내지 10배 부피, 바람직하게는 약 5 내지 10배 부피의 비율로 사용될 수 있다. The amount of the extraction solvent used is not particularly limited. For example, the extraction solvent may be used in an amount of about 1 to 10 times the volume, preferably about 5 to 10 times the volume, based on 1 weight of the Ecklonia cava powder sample.
본 발명에 있어서, 감태 추출물은 감태의 효소 가수분해물, 주정 추출물 및 열수 추출물을 모두 포함한다.In the present invention, the Ecklonia cava extract includes all of the enzyme hydrolyzate, spirit extract and hot water extract of Ecklonia cava.
본 발명에 따라 제조된 모든 감태의 효소 가수분해물, 주정 추출물 및 열수 추출물에서 체지방 감소효과가 확인되었다.Body fat reduction effect was confirmed in the enzyme hydrolyzate, alcohol extract and hot water extract of all Ecklonia cava prepared according to the present invention.
본 발명은 본 발명에 따라 제조된 감태의 효소 가수분해물, 주정 추출물 및 열수 추출물을 유효성분으로 함유하는 약학적 조성물을 제공한다. 상기 감태의 효소 가수분해물, 주정 추출물, 열수 추출물은 바람직하게 약학적 조성물 총 중량에 대하여 0.01 내지 99 중량%를 함유하는 것으로 한다.The present invention provides a pharmaceutical composition containing the enzyme hydrolyzate, alcohol extract and hot water extract of Ecklonia cava prepared according to the present invention as an active ingredient. The enzyme hydrolyzate, ethanol extract, and hot water extract of Ecklonia cava preferably contain 0.01 to 99% by weight based on the total weight of the pharmaceutical composition.
본 발명에 따른 상기 약학적 조성물은 약학적으로 허용 가능한 담체를 포함하여 다양한 형태, 예를 들어 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구용 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제제화될 수 있다. 특히 바람직하게는 경구용 제형으로 제제화될 수 있다.The pharmaceutical composition according to the present invention may be prepared in various forms including pharmaceutically acceptable carriers, for example, oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and the like. It may be formulated in the form of sterile injectable solutions. Especially preferably, it may be formulated in an oral dosage form.
상기 약제학적으로 허용 가능한 담체는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유 등을 포함한다. 또한, 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 포함한다. 경구용 고형 제제는 정제, 환제, 산제, 과립제, 캡슐제 등을 포함하며, 이러한 고형제제는 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트 (calcium carbonate), 수크로오스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 포함할 수 있으며, 마그네슘 스테아레이트, 탈크 같은 윤활제 등을 포함할 수 있다. 경구용 액상 제제는 현탁제, 내용액제, 유제, 시럽제 등을 포함하며, 물, 리퀴드 파라핀 등의 희석제, 습윤제, 감미제, 방향제, 보존제 등을 포함할 수 있다. 비경구용 제제는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제, 좌제를 포함하며, 비수성용제, 현탁제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르류 등을 포함한다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로 젤라틴 등이 사용될 수 있다.The pharmaceutically acceptable carrier is 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, mineral oil and the like. Also included are diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, and the like. Oral solid preparations include tablets, pills, powders, granules, capsules and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, sucrose or lactose. , Gelatin, and the like, and may include a lubricant such as magnesium stearate, talc, and the like. Oral liquid preparations include suspensions, solvents, emulsions, syrups, and the like, and may include water, diluents such as liquid paraffin, wetting agents, sweeteners, fragrances, preservatives, and the like. Parenteral preparations include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories.Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and ethylol. Injectable esters such as late and the like. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycero gelatin and the like can be used.
본 발명은 본 발명에 따라 제조된 감태의 효소 가수분해물, 주정 추출물 및 열수 추출물을 유효성분으로 함유하는 약학적 조성물을 제공한다. 상기 감태의 효소 가수분해물, 주정 추출물, 열수 추출물은 바람직하게 건강기능성식품 조성물 총 중량에 대하여 0.01 내지 99 중량%를 함유하는 것으로 한다.The present invention provides a pharmaceutical composition containing the enzyme hydrolyzate, alcohol extract and hot water extract of Ecklonia cava prepared according to the present invention as an active ingredient. The enzyme hydrolyzate, alcohol extract, and hot water extract of Ecklonia cauliflower should preferably contain 0.01 to 99% by weight based on the total weight of the health functional food composition.
본 발명의 식품 조성물은 건강기능식품으로서 사용될 수 있다. 상기 "건강기능식품"이라 함은 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.The food composition of the present invention can be used as a dietary supplement. The term "health functional food" means a food manufactured and processed using raw materials or ingredients having functional properties useful for the human body according to Act No. 6767 of the Health Functional Food Act, and "functionality" refers to the structure of the human body. And ingestion for the purpose of obtaining nutrients for function or for obtaining useful effects in health uses such as physiological actions.
본 발명의 식품 조성물은 통상의 식품 첨가물을 포함할 수 있으며, 상기 "식품 첨가물"로 서의 적합 여부는 다른 규정이 없는 한, 식품의약품안전청에 승인된 식품 첨가물 공전의 총칙 및 일반시험법 등에 따라 해당 품목에 관한 규격 및 기준에 의하여 판정한다.The food composition of the present invention may include a conventional food additives, and the suitability as the "food additives", unless otherwise specified, in accordance with the General Regulations and General Test Methods of the Food Additives approved by the Food and Drug Administration Judge according to the standards and standards for the item.
상기 "식품 첨가물 공전"에 수재된 품목으로는 예를 들어, 케톤류, 글리신, 구연산칼륨, 니코틴산, 계피산 등의 화학적 합성물, 감색소, 감초추출물, 결정셀룰로오스, 고량색소, 구아검 등의 천연첨가물, L-글루타민산나트륨제제, 면류첨가알칼리제, 보존료제제, 타르색소제제 등의 혼합제제류들을 들 수 있다.Examples of the items listed in the "Food Additive Revolution" include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamon acid, natural additives such as color pigments, licorice extracts, crystalline cellulose, high color pigments, guar gum, And mixed preparations such as sodium L-glutamate, algae additives, preservatives and tar dyes.
캅셀 형태의 건강기능식품 중 경질 캅셀제는 통상의 경질캅셀에 생약 추출물에 부형제 등의 첨가제와의 혼합물 또는 그의 입상물 또는 제피한 입상물을 충진하여 제조할 수 있으며, 연질 캅셀제는 생약 추출물에 부형제 등의 첨가제와의 혼합물을 젤라틴 등 캅셀기제에 충진하여 제조할 수 있다. 상기 연질 캅셀제는 필요에 따라 글리세린 또는 소르비톨 등의 가소제, 착색제, 보존제 등을 함유할 수 있다.Among the health functional foods in the form of capsules, the hard capsules may be prepared by filling a conventional hard capsule with a mixture of additives such as an excipient or the like, or granules or exfoliated granules thereof, and a soft capsule agent with an excipient, etc. It can be prepared by filling a mixture with an additive of a capsule base such as gelatin. The soft capsule agent may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, as necessary.
환 형태의 건강기능식품은 생약 추출물에 부형제, 결합제, 붕해제 등의 혼합물을 적당한 방법으로 성형하여 조제할 수 있으며, 필요에 따라 백당이나 다른 적당한 제피제로 제피를, 또는 전분, 탈크 또는 적당한 물질로 환의를 입힐 수도 있다.The dietary supplement in cyclic form can be prepared by molding a mixture of excipients, binders, disintegrants, etc. in the herbal extracts in a suitable manner, and if necessary, the coating is carried out with white sugar or other suitable coating agent, or with starch, talc or a suitable substance. You may be greeted.
과립형태의 건강기능식품은 상기 생약 추출물에 부형제, 결합제, 붕해제 등의 혼합물을 적당한 방법으로 입상으로 제조할 수 있으며, 필요에 따라 착향제, 교미제 등을 함유할 수 있다. 본원 발명의 상기 부형제, 결합제, 붕해제, 활택제, 교미제, 착향제 등에 대한 용어 정의는 당업계에 공지된 문헌에 기재된 것으로 그 기능 등이 동일 내지 유사한 것들을 포함한다 (대한약전 해설편, 문성사, 한국약학대학협의회, 제 5 개정판, p33-48, 1989).The health functional food in the form of granules may be prepared into granules in a suitable manner by mixing a mixture of an excipient, a binder, a disintegrant, and the like with the herbal extract, and may contain a flavoring agent, a copper, and the like as necessary. The term definitions of the excipients, binders, disintegrants, glidants, copulation agents, flavoring agents, etc. of the present invention are described in documents known in the art and include those having the same or similar functions. Sacrament, Korean College of Pharmacy, 5 revised edition, p33-48, 1989).
이하, 본 발명을 하기 실시예에 의해 더욱 구체적으로 설명한다. 그러나, 이들 실시예는 본 발명에 대한 이해를 돕기 위한 목적일 뿐이므로, 어떤 의미로든 본 발명의 범위가 이들에 의해 제한되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only for the purpose of understanding the present invention, and the scope of the present invention is not limited by them in any sense.
<실시예 1> 본 발명 감태 추출물 제조Example 1 Preparation of Ecklonia cava Extract of the Present Invention
본 발명에 사용한 분말 상태의 감태는 제주특별자치도 서귀포시 대정읍에 위치한 태림상사에서 구입하여 본 발명에 사용하였다.The Ecklonia cava of the powder state used in the present invention was purchased from Taerim Co., Ltd. located in Daejeong-eup, Seogwipo-si, Jeju-do and used in the present invention.
제조예 1: 본 발명 프로텍스 6엘 효소 가수분해 감태추출물Preparation Example 1 Protex 6L Enzymatic Hydrolysis Ecklonia cava Extract
분말상태의 감태 375 g을 pH 7로 조정된 증류수 15 L와 혼합하고, 프로텍스 6엘 효소(130,000 PC/g 이상) 7.5 mL을 첨가하여 60에서 24시간 동안 반응한 다음 그 반응액을 다시 90에서 15분간 추가로 반응시켜 효소 활성을 실활 시켰다. 상기 실활 반응이 끝난 반응액은 3,000 rpm, 4 에서 20분간 원심분리 하였다. 상기 원심분리가 끝난 반응액의 상층액은 여과 및 농축 후 분무건조하여 본 발명 프로텍스 6엘 효소 가수분해 감태추출물을 제조하였고 -18에서 보관하면서 본 발명의 공시시료로 사용하였다.375 g of powdered Ecklonia cava were mixed with 15 L of distilled water adjusted to pH 7, 7.5 mL of Protex 6L enzyme (more than 130,000 PC / g) was added and reacted for 60 to 24 hours. The reaction was further inactivated for 15 minutes at. The deactivation reaction was centrifuged for 20 minutes at 4, 3,000 rpm. The supernatant of the reaction solution after the centrifugation was filtered and concentrated to spray dry to prepare the present invention Protex 6L enzyme hydrolysis Ecklonia cava extract and was used as the test sample of the present invention while kept at -18.
제조예 2: 본 발명 라피다제 프레스 엘 효소 가수분해 감태추출물Preparation Example 2: Rapidase Press L Enzyme Hydrolysis Ecklon Extract of the Present Invention
분말상태의 감태 375 g을 pH 4.5로 조정된 증류수 15 L와 혼합하고, 라피다제 프레스 엘 효소(pectinase 180,000 AVJP/g 이상, cellulase 16,300 CU/g) 7.5 mL을 첨가하여 50에서 24시간 동안 반응한 다음 그 반응액을 3,000 rpm, 4 에서 20분간 원심분리 하였다. 원심분리가 끝난 반응액의 상층액은 여과 및 농축 후 분무건조하여 본 발명 라피다제 프레스 엘 효소 가수분해 감태추출물을 제조하였고 -18에서 보관하면서 본 발명의 공시시료로 사용하였다.375 g of powdered Ecklonia cava were mixed with 15 L of distilled water adjusted to pH 4.5, and 7.5 mL of rapidase pressellase (pectinase 180,000 AVJP / g or more, cellulase 16,300 CU / g) was added and reacted for 50 to 24 hours. The reaction solution was then centrifuged at 3,000 rpm for 4 minutes. The supernatant of the reaction solution after centrifugation was filtered, concentrated and spray-dried to prepare the present invention rapidase press L enzyme hydrolysis Ecklonia cava extract, which was used as the blank sample of the present invention while kept at -18.
제조예 3: 본 발명 로하멘트 씨엘 효소 가수분해 감태추출물Preparation Example 3 Lohament CL enzyme hydrolysis Ecklonia cava extract
분말상태의 감태 375 g을 pH 4.5로 조정된 증류수 15 L와 혼합하고, 로하멘트 씨엘 효소(-glucanase 15,000 ECU/g, cellulase 27,000 CU/g) 7.5 mL을 첨가하여 50에서 24시간 동안 반응한 다음 그 반응액을 3,000 rpm, 4 에서 20분간 원심분리 하였다. 원심분리가 끝난 반응액의 상층액은 여과 및 농축 후 분무건조하여 본 발명 로하멘트 씨엘 효소 가수분해 감태추출물을 제조하였고 -18에서 보관하면서 본 발명의 공시시료로 사용하였다.375 g of powdered Ecklonia cava were mixed with 15 L of distilled water adjusted to pH 4.5, 7.5 mL of Lohment's CI enzyme (-glucanase 15,000 ECU / g, cellulase 27,000 CU / g) was added and reacted for 50 to 24 hours. The reaction solution was centrifuged at 3,000 rpm for 4 minutes. The supernatant of the reaction solution after the centrifugation was filtered, concentrated, and spray dried to prepare the loach cement enzyme hydrolyzed Ecklonia cava extract of the present invention, which was used as the test sample of the present invention while kept at -18.
제조예 4: 본 발명 라피다제 프레스 엘 및 로하멘트 씨엘 효소 가수분해 감태추출물Preparation Example 4 Rapidase Press L and Lohament CL Enzyme Hydrolysis Ecklon Extracts of the Present Invention
분말상태의 감태 375 g을 pH 4.5로 조정된 증류수 15 L와 혼합하고, 라피다제 프레스 엘 효소 및 로하멘트 씨엘 효소(라피다제 프레스 엘 효소: pectinase 180,000 AVJP/g 이상, cellulase 16,300 CU/g)(로하멘트 씨엘 효소:-glucanase 15,000 ECU/g, cellulase 27,000 CU/g) 를 각각 3.75 및 3.75 mL 씩 첨가하여 50에서 24시간 동안 반응한 다음 그 반응액을 3,000 rpm, 4 에서 20분간 원심분리 하였다. 원심분리가 끝난 반응액의 상층액은 여과 및 농축 후 분무건조하여 본 발명 라피다제 프레스 엘 및 로하멘트 씨엘 효소 가수분해(이하 'R+R' 이라 한다) 추출물을 제조하였고 -18에서 보관하면서 본 발명의 공시시료로 사용하였다.375 g of powdered Ecklonia cava were mixed with 15 L of distilled water adjusted to pH 4.5, and the rapidase press L enzyme and the lower halide CI enzyme (rapidase press L enzyme: pectinase 180,000 AVJP / g or more, cellulase 16,300 CU / g) ( Loachment CL enzyme: -glucanase 15,000 ECU / g, cellulase 27,000 CU / g) was added to 3.75 and 3.75 mL respectively to react for 50 to 24 hours and then the reaction solution was centrifuged at 3,000 rpm, 4 for 20 minutes. The supernatant of the reaction solution after centrifugation was filtered, concentrated and spray-dried to prepare extracts of the present invention Rapidase Press L and Lohment CI Enzyme hydrolysis (hereinafter referred to as 'R + R'). It was used as a blank sample of the invention.
제조예 5: 본 발명 감태 80% 발효주정 추출물Preparation Example 5 Ecklonia cava 80% Fermented Alcohol Extract of the Present Invention
분말상태의 감태 375 g을 80% 발효주정 15 L에 침지한 다음 상온에서 24시간 동안 추출하였고 추출이 끝난 추출액을 3,000 rpm, 4 에서 20분간 원심분리 하였다. 원심분리가 끝난 80% 발효주정 추출액은 여과 및 농축 후 분무건조하여 본 발명 감태 80 발효주정 추출물을 제조하였고 -18에서 보관하면서 본 발명의 공시시료로 사용하였다.375 g of powdered Ecklonia cava was immersed in 15 L of 80% fermented alcohol and then extracted at room temperature for 24 hours, and the extracted extract was centrifuged at 3,000 rpm and 4 for 20 minutes. The 80% fermented alcohol extract after centrifugation was filtered, concentrated and spray dried to prepare the Ecklonia cava 80 fermentation extract of the present invention, and was used as the test sample of the present invention while kept at -18.
제조예 6: 본 발명 감태 60% 발효주정 추출물Preparation Example 6 Ecklonia cava 60% Fermented Alcohol Extract of the Present Invention
분말상태의 감태 375 g을 60% 발효주정 15 L에 침지한 다음 상온에서 24시간 동안 추출하였고 추출이 끝난 추출액을 3,000 rpm, 4 에서 20분간 원심분리 하였다. 원심분리가 끝난 60% 발효주정 추출액은 여과 및 농축 후 분무건조하여 본 발명 감태 60 발효주정 추출물을 제조하였고 -18에서 보관하면서 본 발명의 공시시료로 사용하였다.375 g of powdered Ecklonia cava was immersed in 15 L of 60% fermented alcohol and then extracted at room temperature for 24 hours. The extracted extract was centrifuged at 3,000 rpm and 4 for 20 minutes. Centrifuged 60% fermented alcohol extract was filtered and concentrated and spray dried to prepare the Ecklonia cava 60 fermented alcohol extract of the present invention, which was used as the test sample of the present invention while kept at -18.
제조예 7: 본 발명 감태 90 열수 추출물Preparation Example 7 Ecklonia cava 90 hydrothermal extract
분말상태의 감태 375 g을 증류수 15 L와 혼합한 다음 90에서 24시간 동안 열수 추출하였고 열수추출이 끝난 추출액을 3,000 rpm, 4 에서 20분간 원심분리 하였다. 원심분리가 끝난 열수 추출액은 여과 및 농축 후 분무건조하여 본 발명 감태 60 열수 추출물을 제조하였고 -18에서 보관하면서 본 발명의 공시시료로 사용하였다.375 g of powdered Ecklonia cava were mixed with 15 L of distilled water, followed by hot water extraction for 90 to 24 hours, and the extracted hot water was centrifuged at 3,000 rpm and 4 for 20 minutes. Centrifuged hot water extract was filtered and concentrated and spray dried to prepare the Ecklonia cava 60 hot water extract of the present invention, and was used as the test sample of the present invention while kept at -18.
제조예 8: 본 발명 감태 60 열수 추출물Preparation Example 8 Ecklonia cava 60 Hot Water Extract
분말상태의 감태 375 g을 증류수 15 L와 혼합한 다음 60에서 24시간 동안 열수 추출하였고 열수 추출이 끝난 추출액을 3,000 rpm, 4 에서 20분간 원심분리 하였다. 원심분리가 끝난 열수 추출액은 여과 및 농축 후 분무건조하여 본 발명 감태 60 열수 추출물을 제조하였고 -18에서 보관하면서 본 발명의 공시시료로 사용하였다.375 g of powdered Ecklonia cava were mixed with 15 L of distilled water, followed by hot water extraction for 60 to 24 hours, and the extracted hot water was centrifuged at 3,000 rpm and 4 minutes for 20 minutes. Centrifuged hot water extract was filtered and concentrated and spray dried to prepare the Ecklonia cava 60 hot water extract of the present invention, and was used as the test sample of the present invention while kept at -18.
상기 제조예 1 내지 8에 따라 제조된 본 발명 감태 추출물의 추출효율은 하기 표 2에 나타냈다.Extraction efficiency of the Ecklonia cava extract of the present invention prepared according to Preparation Examples 1 to 8 is shown in Table 2 below.
추출효율 측정 결과, 동결건조 및 분무건조를 진행에 따른 추출효율은 동일량의 추출에 대한 수율로는 동결건조보다 분무건조가 3-5% 정도 낮은 수율을 보였지만, 분무건조의 경우 건조시 배관 등에서의 손실을 피할 수 없으며, plant scale 규모의 분무건조를 시행할 경우 소규모의 분무건조 수율보다 높은 수율을 나타내는 것이 일반적이므로 수율면에서 동결건조보다 우위에 있다고 볼 수 있으며, 산업화로 진행할 때 경제적 측면에서도 분무건조가 일반적인 건조방법으로 사료된다. 따라서 동결건조 및 분무건조 방법에 따른 수율의 차이는 없는 것으로 나타났다.As a result of measurement of extraction efficiency, the extraction efficiency according to lyophilization and spray drying showed the yield of 3-5% lower than that of freeze drying in the yield of extraction of the same amount, but in the case of spray drying, It is inevitable that the loss of oil is inevitable, and when plant-scale spray drying is performed, the yield is higher than that of small-scale spray drying. Therefore, it is superior to lyophilization in terms of yield. Spray drying is considered a common method of drying. Therefore, there was no difference in yield according to the lyophilization and spray drying methods.
표 2 본 발명에 따른 감태 추출물의 추출효율
추출물 종류 수율(%)
제조예 1 Protex 21.06
제조예 2 Rapidase 19.08
제조예 3 Rohament 18.66
제조예 4 Rapidase+Rohament (R+R) 21.87
제조예 5 80% 주정 3.24
제조예 6 60% 주정 11.42
제조예 7 90 열수 27.75
제조예 8 60 열수 19.31
TABLE 2 Extraction Efficiency of Ecklonia cava Extract According to the Present Invention
Extract Type yield(%)
Preparation Example 1 Protex 21.06
Preparation Example 2 Rapidase 19.08
Preparation Example 3 Rohament 18.66
Preparation Example 4 Rapidase + Rohament (R + R) 21.87
Preparation Example 5 80% spirit 3.24
Preparation Example 6 60% spirit 11.42
Preparation Example 7 90 hydrothermal 27.75
Preparation Example 8 60 hydrothermal 19.31
<실시예 2> 본 발명 감태 추출물의 성분분석Example 2 Component Analysis of the Ecklonia cava Extract of the Present Invention
실험예 1: 본 발명 감태 추출물의 총 페놀화합물 및 총 플로로탄닌 함량 측정Experimental Example 1: Determination of total phenolic compounds and total phlorotannin content of Ecklonia cava extract of the present invention
총 페놀화합물 함량은 Folin-Denis법(Swain and Hillis, 1959)을 변형하여 측정하였다. 상기 제조예 1내지 8에 따라 제조한 본 발명 감태 추출물을 각각의 추출용매에 용해하여 0.5 mg/mL의 농도로 제조한 시료 0.5 mL을 초순수 증류수 6.5 mL에 각각 가하여 1 N Folin-Ciocalteu`s 용액 0.5 mL과 혼합한 후 3분간 반응시켰다. 상기 반응이 끝나면 무수 탄산나트륨 포화용액 1 mL을 첨가하고 전체가 10 mL가 되도록 초순수 증류수를 가하여 상온에서 1시간 방치시킨 후 UV/visible spectrophotometer로 765 nm에서 흡광도를 측정하였다. The total phenolic compound content was determined by modifying the Folin-Denis method (Swain and Hillis, 1959). Ecklonia cava extract of the present invention prepared according to Preparation Examples 1 to 8 were dissolved in each extraction solvent to prepare a concentration of 0.5 mg / mL. sample 0.5 mL was added to 6.5 mL of ultrapure distilled water, and the mixture was mixed with 0.5 mL of 1N Folin-Ciocalteu`s solution and allowed to react for 3 minutes. After the reaction was completed, 1 mL of anhydrous sodium carbonate solution was added, ultra-distilled distilled water was added so that the whole was 10 mL, and left at room temperature for 1 hour, and then absorbance was measured at 765 nm with a UV / visible spectrophotometer.
Gallic acid를 표준물질로 하여 동일한 방법으로 측정한 다음 얻은 표준곡선으로부터 총 페놀화합물 함량을 정량하였다.Gallic acid was measured as the standard, and the total phenolic compound content was quantified from the obtained standard curve.
총 플로로탄닌 함량은 Folin-Ciocalteu법(Waterman and Mole, 1994)을 변형하여 측정하였다. Phloroglucinol을 표준물질로 하여 동일한 방법으로 측정하여 얻은 표준곡선으로부터 총 플로로탄닌 함량을 정량하였다. 상기 제조예 1내지 8에 따라 제조한 본 발명 감태 추출물을 각각의 추출용매에 용해하여 0.5 mg/mL의 농도로 제조한 시료 0.1 mL을 1 N Folin-Ciocalteu`s 용액 1.0 mL에 각각 가하여 혼합한 다음 3분간 실온 방치하였다. 반응이 끝난 상기 반응액에 20% 무수 탄산나트륨 용액 2 mL을 첨가 혼합하고 45분간 암소에서 방치시킨 후 1,600 g에서 8분간 원심분리하였다. 원심분리한 다음 상층액을 회수하여 UV/visible spectrophotometer로 730 nm에서 흡광도를 측정하여 하기 표 3에 나타냈다. Total phlorotannin content was determined by modifying the Folin-Ciocalteu method (Waterman and Mole, 1994). The total phlorotannin content was quantified from the standard curve obtained by the same method using Phloroglucinol as a standard. Ecklonia cava extract of the present invention prepared according to Preparation Examples 1 to 8 were dissolved in each extraction solvent to prepare a concentration of 0.5 mg / mL. sample 0.1 mL was added to 1.0 mL of 1 N Folin-Ciocalteu`s solution, mixed, and allowed to stand at room temperature for 3 minutes. 2 mL of 20% anhydrous sodium carbonate solution was added and mixed with the reaction solution, and the mixture was left in the dark for 45 minutes and centrifuged at 1,600 g for 8 minutes. After centrifugation, the supernatant was recovered, and the absorbance was measured at 730 nm with a UV / visible spectrophotometer.
표 3 본 발명에 따른 감태 가수분해물 및 발효주정 추출물의 폴리페놀함량
추출물 종류 수율(%) 추출물 원물
총폴리페놀함량(g/mL) 플로로탄닌함량(g/mL) 총폴리페놀 중 플로로탄닌 (%) 총 폴리페놀 함량 (g/g) 플로로탄닌 함량 (g/g)
제조예 1 Protex 21.06 250.34 146.26 58.4 52.72 30.80
제조예 2 Rapidase 19.08 233.07 199.47 85.6 44.48 38.06
제조예 3 Rohament 18.66 293.10 124.13 42.4 54.69 23.16
제조예 4 R+R 21.87 234.32 177.11 75.6 51.25 38.73
제조예 5 80% 주정 3.24 738.37 368.18 49.9 23.89 11.93
제조예 6 60% 주정 11.42 812.17 441.37 54.3 92.78 50.40
제조예 7 90 열수 27.75 221.76 133.97 60.4 61.55 37.18
제조예 8 60 열수 19.31 206.74 174.26 84.3 39.92 33.65
TABLE 3 Polyphenol Content of Ecklonia cava Hydrolyzate and Fermented Alcoholic Extract According to the Present Invention
Extract Type yield(%) extract Original
Total Polyphenol Content (g / mL) Florotannin content (g / mL) Phlorotannin in total polyphenols (%) Total Polyphenol Content (g / g) Phlorotannin content (g / g)
Preparation Example 1 Protex 21.06 250.34 146.26 58.4 52.72 30.80
Preparation Example 2 Rapidase 19.08 233.07 199.47 85.6 44.48 38.06
Preparation Example 3 Rohament 18.66 293.10 124.13 42.4 54.69 23.16
Preparation Example 4 R + R 21.87 234.32 177.11 75.6 51.25 38.73
Preparation Example 5 80% spirit 3.24 738.37 368.18 49.9 23.89 11.93
Preparation Example 6 60% spirit 11.42 812.17 441.37 54.3 92.78 50.40
Preparation Example 7 90 hydrothermal 27.75 221.76 133.97 60.4 61.55 37.18
Preparation Example 8 60 hydrothermal 19.31 206.74 174.26 84.3 39.92 33.65
실험결과, 상기 제조예 1내지 8에 따라 제조한 본 발명 감태 추출물 8종의 총 폴리페놀 함량은 206.74 g/mL 내지 812.17 g/mL로 나타났으며, 감태 60% 주정 추출물(812.17 g/mL)에서 가장 높게 추출되어 유기용매 추출이 폴리페놀을 수득하기에 용이한 추출법으로 사료되나, 감태 원물로부터 획득한 추출물의 수율이 낮아 경제성이 낮다고 판단된다.As a result, the total polyphenol content of the eight kinds of Ecklonia cava extract according to Preparation Examples 1 to 8 was found to be 206.74 g / mL to 812.17 g / mL, Ecklonia cava extract 60% (812.17 g / mL) Extraction of organic solvents is the most effective extraction method in order to obtain polyphenols, but the yield of extracts obtained from Ecklonia cava extract is low and economic efficiency is low.
상기 제조예 1내지 8에 따라 제조한 본 발명 감태 추출물중 발효주정 추출물(제조예 5 및 제조예 6)을 제외한 추출물 중 플로로탄닌 함량이 높은 감태 추출물로는 Rapidase 효소 추출물 Rapidase+Rohament 복합효소 추출물 60 열수 추출물 순으로 나타났다. Among the extracts of the present invention prepared according to Preparation Examples 1 to 8 above, except for the fermentation spirit extract (Preparation Example 5 and Preparation Example 6), the extract of Ecklonia cava extract having a high content of phlorotannin is Rapidase enzyme extract Rapidase + Rohament complex enzyme extract. 60 hydrothermal extracts were found in this order.
<실시예 3> 본 발명 감태 추출물의 항비만 효능 검정Example 3 Anti-obesity Effect Assay of Ecklonia cava Extract
실험예 2: 본 발명 감태 추출물의 지방합성 억제 효능검정Experimental Example 2: Test for the inhibition of fat synthesis of Ecklonia cava extract of the present invention
3T3-L1 지방세포 분화 유도Induces 3T3-L1 Adipocyte Differentiation
본 발명에 사용된 3T3-L1 pre-adipocyte 세포는 미국세포주은행 (American Type Culture Collection, CL-173)으로부터 구입하였다. 세포는 10% Fetal Bovine Serum (FBS)가 포함된 Dulbecco's modified Eagle's medium (DMEM)를 사용하여 배양하였다. 3T3-L1 pre-adipocyte는 세포가 다 자라면, 증식을 멈추고 분화가 유도되는 특징을 가지고 있어, 분화유도인자를 첨가하여 지방세포 관련한 실험을 하기에 적합한 세포 모델로 알려져 있다. 3T3-L1 세포가 post-confluent되면 (D-0), 10% FBS-DMEM에 분화유도인자인 0.5mM Methylisobutylxanthine (M, I-5879), 0.25M dexamethasone (D, D-4902), 10g/ml insulin (I, I-1882)을 첨가하여 초기분화를 유도하였다. 또한 D-2, D-4, D-6일째에는 10g/ml insulin만을 첨가하여 지방세포로 분화를 유도하였다. 3T3-L1 pre-adipocyte cells used in the present invention were purchased from the American Cell Line Bank (American Type Culture Collection, CL-173). Cells were cultured using Dulbecco's modified Eagle's medium (DMEM) containing 10% Fetal Bovine Serum (FBS). 3T3-L1 pre-adipocytes are known to be suitable cell models for adipocyte-related experiments by adding differentiation-inducing factors, since they proliferate and induce differentiation when cells mature. When 3T3-L1 cells were post-confluent (D-0), 0.5mM Methylisobutylxanthine (M, I-5879), 0.25M dexamethasone (D, D-4902), 10g / ml Insulin differentiation was induced by the addition of insulin (I, I-1882). On the D-2, D-4, and D-6 days, only 10g / ml insulin was added to induce differentiation into adipocytes.
본 발명에 따른 감태 추출물의 Triglyceride (TG) 함량 감소효과 Triglyceride (TG) Content Reduction Effect of Ecklonia cava Extract According to the Present Invention
분화를 유도한 3T3-L1 adipocyte에 상기 제조예 1내지 8에 따라 제조한 본 발명 감태 추출물을 각각 처리하여 TG(중성지방)의 함량을 측정하여, 중성지방 함량 감소효과를 평가하였다.Differentiation-induced 3T3-L1 adipocytes were treated with the Ecklonia cava extract according to Preparation Examples 1 to 8, respectively, to measure the content of TG (triglycerides), and the triglyceride content reduction effect was evaluated.
3T3-L1 세포에 분화촉진인자를 처리하여 분화를 유도한 뒤에, Serum-free medium (SFM)을 24시간 처리한 다음 상기 제조예 1 내지 4에 따라 제조한 본 발명 감태 추출물 시료 각각을 250, 500g/mL 농도별로 24시간 처리하였다. 시료처리 24시간 후에 상기 시료가 처리된 각각의 세포를 PBS로 두 번 세척하고 ice-cold PBS 500 첨가한 다음에 30초간 초음파 분쇄하여 세포막 안의 내용물이 용출되도록 하였다. 상기 초음파 분쇄하여 수득한 Cell lysate의 TG 함량은 아산제약의 TG 측정 시약을 사용하여 분석하였다. After inducing differentiation by treating differentiation promoting factors on 3T3-L1 cells, Serum-free medium (SFM) was treated for 24 hours, followed by 250, 500 g of each of the Ecklonia cava extract samples prepared according to Preparation Examples 1 to 4 above. Treatment was performed for 24 hours at / mL concentration. After 24 hours of sample treatment, each cell treated with the sample was washed twice with PBS, added with ice-cold PBS 500, and sonicated for 30 seconds to allow the contents in the cell membrane to be eluted. TG content of the cell lysate obtained by the ultrasonic grinding was analyzed using TG measuring reagent of Asan Pharmaceutical.
상기 cell lysate 10에 TG assay reagent 200를 첨가하여 혼합한 다음에 37에서 10분간 반응시킨 후 multi-well plate reader를 사용하여 550nm 흡광도에서 측정하였다. TG assay reagent 200 was added to the cell lysate 10, mixed, and then reacted at 37 for 10 minutes, and then measured at 550 nm absorbance using a multi-well plate reader.
실험결과, 도 1에 나타낸 바와 같이 상기 제조예 1 내지 4에 따라 제조된 모든 감태 추출물 처리에 의해서 지방세포분화가 유도된 MDI 군과 비교하여 중성지방의 함량이 감소한 것으로 나타났다. As a result, as shown in FIG. 1, the content of triglycerides was reduced in comparison with the MDI group in which adipocyte differentiation was induced by all the Ecklonia cava extract preparations prepared according to Preparation Examples 1 to 4.
상기 제조예 1 내지 4를 처리하여 중성지방 함량 감소효과를 확인한 결과에서 알 수 있듯이 250g/mL의 농도에서도 500g/mL의 농도와 유사한 효과를 보였기 때문에 세포 처리농도를 250g/mL농도로 결정하였다. As shown in the results of confirming the effect of reducing the triglyceride content by treating the Preparation Examples 1 to 4, the cell treatment concentration was determined as 250g / mL concentration because it showed a similar effect to the concentration of 500g / mL even at the concentration of 250g / mL.
또, 분화를 유도한 3T3-L1 adipocyte에 상기 제조예 3내지 8에 따라 제조한 본 발명 감태 추출물을 각각 처리하여 TG(중성지방)의 함량을 측정하여, 중성지방 함량 감소효과를 평가하였다. 실험 방법은 상기 제조예 1 내지 4에 따라 제조된 감태 추출물들의 중성지방함량 감소효과 측정방법과 동일하게 진행하였고 처리 농도는 추출물별로 각각 12.5, 50, 200 g/mL로 하여 처리하였다.In addition, by treating each of the Ecklonia cava extract prepared according to Preparation Examples 3 to 8 to differentiation-induced 3T3-L1 adipocyte, the content of TG (triglyceride) was measured to evaluate the effect of reducing triglyceride content. Experimental method was carried out in the same manner as the method for measuring the triglyceride content reduction effect of the Ecklonia cava extract prepared according to Preparation Examples 1 to 4 and the treatment concentration was treated to 12.5, 50, 200 g / mL for each extract.
실험결과, 도 2에 나타낸 바와 같이 지방세포내의 중성지방의 함량은 상기 제조예 3 내지 8에 따라 제조된 추출물에 의해서 모두 감소하는 효과가 나타났다.As a result, as shown in FIG. 2, the content of triglycerides in adipocytes was reduced by the extract prepared according to Preparation Examples 3 to 8.
상기 본 발명 제조예 1 내지 8에 따라 제조된 감태 가수분해물 및 발효주정 추출물 모두에서 지방세포의 중성지방함량 감소 효과가 나타났다. The effect of reducing triglycerides content of adipocytes was observed in both the Ecklonia cava hydrolyzate and fermented alcohol extract prepared according to Preparation Examples 1 to 8 of the present invention.
따라서, 이하, 하기에 실시된 모든 실시예 및 실험예에서는 제조예 4 및 제조예 8에 따라 제조된 감태 가수분해물과 발효주정 추출물로서 체지방 감소효과를 평가 하였으나, 하기 실시예 및 실험예의 결과는 본 발명 제조예 1내지 8에 이르는 모든 감태 가수분해물 및 발효주정 추출물의 효과임이 자명하다.Therefore, in the following Examples and Experimental Examples all carried out below to evaluate the effect of reducing body fat as the Ecklonia hydrolyzate and fermented alcohol extract prepared according to Preparation Examples 4 and 8, the results of the following Examples and Experimental Examples It is apparent that the effects of all the Ecklonia cava hydrolyzate and fermented alcoholic extracts of the inventive preparations 1 to 8 are effective.
실험예 3: 본 발명 감태 추출물의 세포생존율 검정Experimental Example 3: Cell viability assay of the Ecklonia cava extract of the present invention
세포 생존율 측정에 사용된 시료는 체중감소에 효과가 있다고 종래에 보고된 가르시니아 추출물 및 플로로탄닌 기본구조체인 phloroglucinol (ALDRICH 79330)을 양성대조군으로 각각 설정하고, 상기 제조예 4(R+R 효소 추출물), 제조예 8(60 열수추출물)에 따라 제조된 시료의 세포생존율 검정을 수행하였다. Samples used for measuring cell viability are effective in weight loss, and the conventionally reported garcinia extract and phloroglucinol (ALDRICH 79330), a phloroglucin basic structure, were set as positive controls, respectively, and Preparation Example 4 (R + R enzyme extract). ), Cell viability assay of the sample prepared according to Preparation Example 8 (60 hot water extract).
가르시니아 추출물, phloroglucinol, 상기 제조예 4, 제조예 8에 따라 제조된 시료를 각각 농도별로 처리한 세포의 생존율의 변화를 확인하고자, CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay kit (promega, G5421)를 사용하여 세포 생존율을 측정하였다.To determine the change in viability of cells treated with garcinia extract, phloroglucinol, and the samples prepared according to Preparation Examples 4 and 8, respectively, by concentration, CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay kit (promega, G5421) was used. Cell viability was measured.
96-well plate에 24 cells/well의 농도로 3T3-L1 세포를 분주(seeding)하고 20시간 동안 배양하였다. 배양을 마친 각 세포에 SFM을 4시간 처리한 다음 가르시니아 추출물 (G), Phloroglucinol (P), 제조예 4(Rapidase + Rohament; R+R), 제조예 8(60 열수추출물; 60 H) 시료 각각을 12.5 50, 200g/mL의 농도별로 세포에 처리한 다음 24 및 48시간 동안 배양하였다. 배양이 끝난 세포의 배지를 제거하고, phenol-red free DMEM에 MTS/PMS solution을 첨가하여 30분 동안 배양한 후에 multi-well plate reader를 사용하여 490nm의 흡광도에서 측정하였다. 음성대조군(CON)군의 세포생존율과 비교하여 분석하였다. 3T3-L1 cells were seeded at a concentration of 2 4 cells / well in a 96-well plate and incubated for 20 hours. Each cell was treated with SFM for 4 hours, followed by Garcinia extract (G), Phloroglucinol (P), Preparation Example 4 (Rapidase + Rohament; R + R), and Preparation Example 8 (60 hydrothermal extract; 60 H). The cells were treated with cells at concentrations of 12.5 50 and 200 g / mL and then incubated for 24 and 48 hours. After culturing, the culture medium was removed, and MTS / PMS solution was added to phenol-red free DMEM, followed by incubation for 30 minutes, and the absorbance was measured at 490 nm using a multi-well plate reader. The cell survival rate of the negative control group (CON) group was analyzed.
실험결과, 도 3에 나타낸 바와 같이 3T3-L1 세포에 제조예 4(R+R) 및 제조예 8(60 H) 감태 추출물을 처리하였을 때, 모든 실험군에서 음성대조군(COM)과 비교했을 때 세포가 증식하는 것으로 나타났으며, 24시간 처리 시 세포 증식율이 더 높은 것으로 나타났다.  As a result of the experiment, when 3T3-L1 cells were treated with Preparation Example 4 (R + R) and Preparation Example 8 (60H) Ecklonia cava extract as shown in FIG. 3, the cells were compared with the negative control group (COM) in all experimental groups. Was found to proliferate, and cell proliferation was higher at 24 h treatment.
또, 양성대조군인 Phloroglucinol 처리 시 농도에 따라 세포 생존율이 감소하는 경향을 보였으나, 음성대조군(CON) 수준 이상에서 감소하는 경향을 보여 시료 처리에 따른 세포 생존율의 감소는 보이지 않았다. 따라서 이후의 실험에서는 24시간을 기준으로 실험을 진행하였다.   In addition, the cell survival rate decreased with the concentration of the positive control Phloroglucinol, but showed a tendency to decrease above the negative control (CON) level, did not show a decrease in the cell survival rate according to the sample treatment. Therefore, in the subsequent experiment, the experiment was conducted based on 24 hours.
실험예 4: 본 발명 감태 추출물에 따른 지방대사관련 단백질 발현변화 분석Experimental Example 4: Analysis of changes in protein expression related to fat metabolism according to the Ecklonia cava extract
본 발명에 따라 제조된 8종의 감태 추출물들의 중성지방 함량 감소 효과(실험예 2)와 세포생존율 증대효과(실험예 3)를 통하여 확인한 바와 같이 8종의 추출물이 모두 유사한 활성을 갖는다. 따라서 상업적으로 이용할 때 경제적이라고 사료되는 제조예 4 및 8에 따라 제조된 감태 추출물만을 본 실험예의 시료로 사용하였다. Eight extracts have a similar activity as confirmed through the triglyceride content reduction effect (Experimental Example 2) and cell viability increase effect (Experimental Example 3) of the eight kinds of Ecklonia cava extract prepared according to the present invention. Therefore, only the Ecklonia cava extract prepared according to Preparation Examples 4 and 8, which is considered economical when used commercially, was used as a sample of this Experiment.
상기 제조예 4 및 제조예 8에 따라 제조된 감태 추출물의 지방대사 관련 단백질의 발현에 미치는 영향을 확인하고자, western blotting을 수행하였다. In order to confirm the effect on the expression of lipo metabolism-related protein of Ecklonia cava extract prepared according to Preparation Example 4 and Preparation Example 8, western blotting was performed.
6-well plate에 3T3-L1 세포를 분주(seeding)하고 80% confluent되면, 분화촉진인자를 처리하여 분화를 6일 동안 유도하였다. 분화유도한 다음 각 세포의 배양배지를 제거한 다음 Serum-free medium (SFM)로 교체하여 24시간 동안 무혈청 배지조건으로 세포를 배양한다. 무혈청배지상태에 있는 세포에 50g/mL의 가르시니아 추출물(G)과 Rapidase + Rohament (R+R; 제조예 4), 60 열수추출물 (60 H;제조예 8) 각각의 시료를 12.5 50, 200g/mL의 농도별로 24시간 처리하였다. Differentiation was induced for 6 days by seeding 3T3-L1 cells in a 6-well plate and processing 80% confluent. After induction of differentiation, the culture medium of each cell is removed and replaced with Serum-free medium (SFM) to incubate cells in serum-free medium for 24 hours. 50 g / mL Garcinia extract (G), Rapidase + Rohament (R + R; Preparation 4), 60 hydrothermal extracts (60 H; Preparation 8) were added to the cells in serum-free medium. Treatment was performed for 24 hours at a concentration of / mL.
상기 각각의 시료를 24시간 처리한 다음 세포의 배양배지를 제거하고 3T3-L1 세포를 PBS로 두 번 세척한 다음 lysis buffer (20mM Tris, pH 8.0, 150mM NaCl, 10mM sodium phosphate, 100M sodium orthvanadate, 100M ammonium molybdate, 1mM -glycerophosphate, 10% glycerol, 0.1% NP-40, 0.1% SDS, phosphate inhibitor, protease inhibitor)를 각 세포마다 첨가하여 세포를 용해한 다음 그 세포 용해물을 회수하였다. 상기 회수한 각각의 세포 용해물을 단백질 정량을 수행한 다음 동량의 단백질을 815% gel에 각각 전기영동하여 PVDF membrane에 전이시켰다. 상기 각각의 세포의 단백질 용해물이 전이된 PVDF membrane을 1% BSA-TBS-T로 1시간 동안 blocking한 다음 blocking된 PVDF membrane에 분석하고자 하는 각각의 단백질의 1차 항체(1:1000, 1% BSA-TBS-T)를 10에서 16시간 반응시킨 다음 TBS-T로 washing하였다. 상기 washing된 PVDF membrane에 2차 항체 (1:10000)를 각각 붙이고, Chemiluminescent sensitive HRP를 붙여 KODAK X-ray 및 X-OMAT 필름으로 현상하였다. 그 결과는 도 4 내지 도 5에 나타냈다. After each sample was treated for 24 hours, the cell culture medium was removed, and 3T3-L1 cells were washed twice with PBS, followed by lysis buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 10 mM sodium phosphate, 100M sodium orthvanadate, 100M). ammonium molybdate, 1 mM -glycerophosphate, 10% glycerol, 0.1% NP-40, 0.1% SDS, phosphate inhibitor, and protease inhibitor) were added to each cell to lyse the cells, and the cell lysate was recovered. Each of the recovered cell lysates was subjected to protein quantification, and then the same amount of protein was electrophoresed on 815% gel to transfer to PVDF membrane. The PVDF membrane to which the protein lysate of each cell was transferred was blocked with 1% BSA-TBS-T for 1 hour, and then the primary antibody (1: 1000, 1%) of each protein to be analyzed on the blocked PVDF membrane. BSA-TBS-T) was reacted at 10 to 16 hours and then washed with TBS-T. Secondary antibodies (1: 10000) were attached to the washed PVDF membrane, respectively, and developed with KODAK X-ray and X-OMAT films with Chemiluminescent sensitive HRP. The results are shown in FIGS. 4 to 5.
본 발명에 따라 제조된 감태 추출물들의 지방세포 분화억제 효과를 확인하기 위해서 본 실험예에서 분석한 단백질은 대사 증후군의 지표로 사용되는 Acrp30, 에너지 항상성 유지 및 체중조절에 관여하는 Ob (leptin), 지방세포 분화와 지방산 대사조절에 관여하는 PPAR 및 C/EBP, 지방세포분화 표지마커인 aP2, 콜레스테롤, 지방산 및 중성지방 대사에 관련된 유전자들의 발현을 조절하는 SREBP-1, 지방산생성 기작에 관여하는 FAS, LPL 및 pACC- 단백질 등이다. 상기 단백질들의 발현 분석결과는 도 4에 나타냈다.In order to confirm the effect of inhibiting adipocyte differentiation of the Ecklonia cava extracts prepared according to the present invention, the protein analyzed in this example was Acrp30, which is used as an indicator of metabolic syndrome, Ob (leptin), fat involved in maintaining energy homeostasis and weight control. PPAR and C / EBP involved in cell differentiation and fatty acid metabolism regulation, aP2, a marker for adipocyte differentiation, SREBP-1, which regulates the expression of genes related to cholesterol, fatty acid and triglyceride metabolism, FAS involved in fatty acid production mechanism, LPL and pACC-proteins. Expression analysis results of the proteins are shown in FIG.
발현 분석결과, 3T3-L1 지방세포에 본 발명 제조예 4 및 제조예 8에 따라 제조된 감태 추출물 처리시 MDI 처리군에서 Ob 단백질의 발현이 증가하였으며, R+R(제조예 4) 처리군과 60 H(제조예 8) 처리군에서 농도 의존적으로 발현이 감소되었다. As a result of expression analysis, 3T3-L1 adipocytes treated with Ecklonia cava extract prepared according to Preparation Examples 4 and 8 of the present invention increased the expression of Ob protein in the MDI treatment group, and R + R (Preparation Example 4) treatment group and Expression was reduced in a concentration dependent manner in the 60 H (Preparation Example 8) group.
Acrp30은 3T3-L1 지방세포에 본 발명 제조예 4 및 제조예 8에 따라 제조된 감태 추출물 처리 시 감태 추출물 처리농도 의존적으로 발현이 증가하였으나, 지방세포 분화 및 지방생성에 직접적으로 관여하는 단백질 PPAR, C/EPB, SREBP-1, FAS, LPL의 발현은 본 발명 제조예 4 및 제조예 8에 따라 제조된 감태 추출물 처리농도 의존적으로 감소였다. Acrp30 increased the expression of Ecklonia cava extract concentrations according to Ecklonia cava extracts prepared according to Preparation Examples 4 and 8 in 3T3-L1 adipocytes, but the protein PPAR directly involved in adipocyte differentiation and adipogenesis. The expression of C / EPB, SREBP-1, FAS, LPL was reduced depending on the concentration of Ecklonia cava extract prepared according to Preparation Examples 4 and 8 of the present invention.
ACC는 serin 잔기의 인산화 (불활성화) 및 탈인산화 (활성화)에 따라 활성을 조절받는 단백질로서 지방세포에 본 발명 감태 추출물들(제조예 4 및 제조예 8)을 농도별로 처리하였을 때 ACC-의 인산화 수준이 농도 의존적으로 감소하였다.ACC is a protein whose activity is regulated according to phosphorylation (inactivation) and dephosphorylation (activation) of serin residues, and the concentration of ACC- when Ecklonia cava extracts (Preparation Example 4 and Preparation Example 8) were treated in adipocytes by concentration. Phosphorylation levels decreased concentration dependently.
따라서 본 발명에 따라 제조된 감태 추출물들은 지방세포 분화 및 지방생성을 억제하는 뛰어난 효과가 있는 것으로 사료된다.Therefore, the Ecklonia cava extract prepared according to the present invention is considered to have an excellent effect of inhibiting adipocyte differentiation and adipogenesis.
또, 본 발명에 따라 제조된 감태 추출물들의 사이토카인 신호 및 에너지대사 활성화 효과를 확인하기 위해서 본 실험예에서 분석한 단백질은 사이토카인 신호전달을 조절하는 SOCS-3, EGF와 같은 성장 호르몬에 의해 활성화되는 Stat3 및 인산화 Stat3, 체온조절 및 당 대사에 관여하는 UCP2, 당 수송 단백질인 GLUT4 단백질 등이다. 상기 단백질들의 발현 분석결과는 도 5에 나타냈다.In addition, to confirm the cytokine signal and energy metabolic activation effect of the Ecklonia cava extract prepared according to the present invention, the protein analyzed in this experiment is activated by growth hormones such as SOCS-3 and EGF that regulate cytokine signaling. Stat3 and phosphorylated Stat3, UCP2 involved in thermoregulation and sugar metabolism, GLUT4 protein is a sugar transport protein. Expression analysis results of the proteins are shown in FIG.
본 발명 제조예 4 및 제조예 8에 따라 제조된 감태 추출물을 농도별로 3T3-L1 지방세포에 처리하였을 때 Supperssor of cytokine signaling-3 (SOCS-3), Stat3 단백질 및 인산화 Stat3, UCP2의 발현은 감소되었으나, Glut4의 발현에는 영향을 주지 않는 것으로 나타났다. Expression of Supperssor of cytokine signaling-3 (SOCS-3), Stat3 protein and phosphorylated Stat3, UCP2 was reduced when Ecklonia cava extracts prepared according to Preparation Examples 4 and 8 of the present invention were treated to 3T3-L1 adipocytes by concentration. However, it did not appear to affect the expression of Glut4.
<실시예 4> 본 발명 감태 추출물의 항비만효과 검정<Example 4> Anti-obesity effect assay of the present invention Ecklonia cava extract
본 발명 제조예 4 및 제조예 8에 따라 제조된 감태 추출물의 항비만 활성 검정은 C57BL/6 mouse를 10주간의 고지방식이를 통하여 사육한 동물을 이용하여 수행하였다. Anti-obesity activity assay of Ecklonia cava extract prepared according to Preparation Example 4 and Preparation Example 8 of the present invention was carried out using an animal in which C57BL / 6 mouse was bred through a high-fat diet for 10 weeks.
본 발명에 따라 제조된 감태 추출물들(R+R 효소추출물; 제조예 4, 감태 60 열수추출물; 제조예 8)의 항비만 효과를 확인하기 위하여 각각의 시료는 하기 표 5에 나타낸 바와 같이 동물용 사료에 농도별로 첨가하여 동물에게 식이토록 하였다.양성대조군으로 가르시니아 추출물을 사용하여 본 발명 감태 추출물들의 효과와 비교하였다. 실험식이의 조성은 하기 표 4에 제시하였다. In order to confirm the anti-obesity effect of the Ecklonia cava extracts prepared according to the present invention (R + R enzyme extract; Preparation Example 4, Ecklonia cava 60 hydrothermal extract; Preparation Example 8), each sample was used for animals as shown in Table 5 below. It was added to the feed by concentration to the animals. The garcinia extract was used as a positive control to compare the effects of the Ecklonia cava extract of the present invention. The composition of the experimental diet is shown in Table 4 below.
표 4 본 발명에 따른 동물실험용 식이 조성
ND HD HD + G 25 HD
(60H or R+R)+ 5 (60H or R+R) + 25 (60H or R+R) + 150
casein 200 200 200 200 200 200
DL-methionine 3 3 3 3 3 3
sucrose 500 500 500 500 500 500
corn starch 150 150 150 150 150 150
cellulose 50 50 50 50 50 50
corn oil 50 50 50 50 50 50
mineral mix 35 35 35 35 35 35
vitamin mix 10 10 10 10 10 10
choline bitartate 2 2 2 2 2 2
lard 170 170 170 170 170
cholesterol 10 10 10 10 10
sample 6.7 1.3 6.7 40.4
total (g) 1000 1180 1186.7 1181.3 1186.7 1220.4
carlorie 3850 5380 5380 5380 5380 5380
fat 450 1980 1980 1980 1980 1980
fat (% calorie) 11.7 36.8 36.8 36.8 36.8 36.8
Table 4 Dietary composition for animal experiments according to the present invention
ND HD HD + G 25 HD
(60H or R + R) + 5 (60H or R + R) + 25 (60H or R + R) + 150
casein 200 200 200 200 200 200
DL-methionine 3 3 3 3 3 3
sucrose 500 500 500 500 500 500
corn starch 150 150 150 150 150 150
cellulose 50 50 50 50 50 50
corn oil 50 50 50 50 50 50
mineral mix 35 35 35 35 35 35
vitamin mix 10 10 10 10 10 10
choline bitartate 2 2 2 2 2 2
lard 170 170 170 170 170
cholesterol 10 10 10 10 10
sample 6.7 1.3 6.7 40.4
total (g) 1000 1180 1186.7 1181.3 1186.7 1220.4
carlorie 3850 5380 5380 5380 5380 5380
fat 450 1980 1980 1980 1980 1980
fat (% calorie) 11.7 36.8 36.8 36.8 36.8 36.8
정상식이 (Normal Diet, ND)는 AIN-76 semipurified diet (MP 0290545220)조성에 준하여 조제되었고 (corn oil, 5% wt/wt), 고지방식이 (High Fat Diet, HFD)는 lard와 corn oil (17: 5)를 지질급원으로 사용하였으며, 식이 내 지방함량이 20% (총 열량의 37%) 수준이 되도록 조정하였다. 다른 선행연구들에 의하면 식이지방을 20% wt/wt 수준으로 첨가 시 총 열량의 40%를 지방급원으로 공급받게 되며, 이는 20% 정도를 지질에서 공급받는 한국인의 식생활패턴과 비교하여 고지방식이로 인한 비만모델에 적합한 수준으로 판단되어 실험을 수행하였다. 실험식이 제조에 필요한 시료는 가르시니아 섭취량을 동물에 적용한 결과, 25mg/20kcal/day로 계산됨에 따라, 감태 시료는 5mg, 25mg, 150mg/20kcal/day이 되도록 사료를 제조하였다. The Normal Diet (ND) was formulated according to the AIN-76 semipurified diet (MP 0290545220) (corn oil, 5% wt / wt) and the high fat diet (HFD) was lard and corn oil ( 17: 5) was used as a lipid source, and the dietary fat content was adjusted to 20% (37% of total calories). According to other previous studies, when dietary fat is added at 20% wt / wt level, 40% of total calories are supplied to fat source, which is about 20% compared to the dietary patterns of Koreans fed from lipids. The experiment was conducted as judged to be suitable for the obesity model. As the sample required for the preparation of the experimental diet was applied to the animal ingestion of garcinia was calculated as 25mg / 20kcal / day, the Ecklonia cava sample was prepared to be 5mg, 25mg, 150mg / 20kcal / day.
실험예 5: 본 발명 감태 추출물처리에 따른 마우스의 체중감소 효과Experimental Example 5: Weight loss effect of the mouse according to the present invention Ecklonia cava extract treatment
사료섭취량은 매일 측정하여 기록하였으며, 체중증가량은 7일에 1회씩 측정하였다. 도 6에 나타냈다.Feed intake was measured and recorded daily, and weight gain was measured once every 7 days. 6 is shown.
실험결과, 동물실험시작시 전체 마우스의 평균 체중은 20.8 0.44g이었으며, 10주 사육종료 후 체중 변화는 정상식이군 (ND) 32.0, 고지방식이 대조군 (HD) 36.2, 가르시니아 추출물식이군 (GAR 25) 32.5, 감태 60 열수추출물식이군 (E. cava 60H) 34.2, 36.3, 30.6, 감태 R+R 효소처리식이군 (E. cava R+R) 36.2, 33.6, 26.2으로 감태 실험식이의 농도에 따라 체중이 감소하였다. As a result, the average body weight of the mice at the start of animal experiment was 20.8 0.44g, and the change in body weight after 10 weeks of breeding was normal diet (ND) 32.0, high fat diet (HD) 36.2, garcinia extract diet (GAR 25). 32.5, Ecklonia cava 60H (E. cava 60H) 34.2, 36.3, 30.6, Ecklonia cava R + R Enzyme-treated diet (E. cava R + R) 36.2, 33.6, 26.2 Decreased.
특히, 감태 60 열수추출물 150mg(제조예 8)군과 감태 R+R 효소처리(제조예 4) 150mg군에서는 정상식이군과 비교했을 때 유의적으로 체중이 감소하였다. In particular, weight loss was significantly decreased in 150 mg of Ecklonia 60 hot water extract (Preparation Example 8) and 150 mg of Ecklonia cava R + R enzyme treatment (Preparation Example 4) compared to the normal diet group.
실험예 6: 본 발명 감태 추출물처리에 따른 고지방식이 마우스의 간 및 지방 무게 변화분석Experimental Example 6 Analysis of Liver and Fat Weight Changes in High Fat Diet Mice Treated with Ecklonia cava Extract
10주 사육 종료 후 마우스를 하루 절식시킨 다음 diethyl ether로 마취한 상태에서 간, 부고환지방, 신장주변의 지방을 적출하여 무게를 측정하였다.After 10 weeks of breeding, mice were fasted for one day, and liver, epididymal fat, and fat from the kidneys were extracted under anesthesia with diethyl ether.
실험결과, 도 7에 나타낸 바와 같이 간 조직의 무게는 앞선 체중의 결과와 동일한 경향을 보여주었다. 감태 60 열수추출물 150mg군과 감태 R+R 효소처리 150mg군에서는 정상식이군과 비교했을 때 유의적으로 간 조직의 무게가 감소하는 것으로 나타났다. As a result, as shown in Figure 7, the weight of liver tissue showed the same tendency as the result of the previous weight. The weight of liver tissue was significantly decreased in the 150 mg group of Ecklonia cava 60 hot water extract and the 150 mg Ecklonia cava R + R enzyme treatment group compared with the normal diet group.
부고환 지방의 무게는 도 8에 나타낸 바와 같이 본 발명 감태 열수추출물 150mg 식이군과 R+R 효소처리 150mg 식이군에서는 정상식이군과 비슷한 수준의 무게까지 감소하였다. 그러나 도 9에 나타난 바와 같이 신장주변의 지방무게에 있어서는 본 발명 감태 추출물들의 처리에 의한 유의적인 감소는 나타나지 않았다.As shown in FIG. 8, the weight of epididymal fat was reduced to the level of weight similar to that of the normal diet group in the 150 mg diet group of the Ecklonia cava hot-water extract and the R + R enzyme-treated 150 mg diet group. However, as shown in Figure 9 in the fat weight around the kidney did not show a significant decrease by the treatment of the Ecklonia cava extract of the present invention.
실험예 7: 본 발명 감태 추출물처리에 따른 고지방식이 마우스의 혈액학적 변화분석Experimental Example 7: Analysis of hematological changes in high fat diet mice according to the present invention Ecklonia cava extract treatment
10주 사육 종료 후 마우스를 하루 절식시킨 다음 diethyl ether로 마취한 상태에서 단두하여 혈액을 회수하고 그 혈액을 2,500 rpm에서 20분간 원심분리하여 혈청을 분리하여 혈청 중의 당, Cholesterol, HDL-cholesterol, tiglyceride 및 leptin 농도를 측정하였다. After 10 weeks of breeding, mice were fasted for one day, and then anesthetized with diethyl ether to collect blood, and the blood was centrifuged at 2,500 rpm for 20 minutes to separate serum, and sugar, Cholesterol, HDL-cholesterol and tiglyceride in serum. And leptin concentrations were measured.
혈당분석Blood sugar analysis
감태 추출물을 10주간 투여 후, 최종 부검일에 채혈을 통해 얻어진 혈청을 혈당 측정 kit(아산제약, AM 201-K)을 이용하여 마우스의 혈중 GLUCOSE의 농도를 측정하였으며, 효소시액 3ml을 기준으로 표준액을 통해 정량선을 작성한 후, 냉동 보관된 혈청 중 20ul을 취하여 분광광도계에서 500nm에서 측정하였다.After administration of Ecklonia cava extract for 10 weeks, the serum obtained from blood collection on the final autopsy day was measured using a blood glucose measurement kit (Asan Pharmaceutical Co., AM 201-K) to measure the concentration of GLUCOSE in mice. After making a determination line through, 20ul of frozen serum was taken and measured at 500nm with a spectrophotometer.
실험결과, 도 10에 나타난 바와 같이 정상식이군(ND)과 비교하여 고지방식이군(HD)에서 혈당이 유의적으로 증가하였고, 양성대조군으로 사용된 가르시니아군(GAR 25), 감태 효소처리군 (R+R 150), 및 감태 열수추출물 150mg (60H 150) 처리군에서 유의적으로 혈중 혈당량이 감소하였다. As a result, as shown in FIG. 10, the blood sugar level was significantly increased in the high-fat diet group (HD) compared to the normal diet group (ND), and the garcinia group (GAR 25) used as a positive control group, and the Eckonia enzyme treatment group (R + R 150), and EEG 150 mg (60H 150) treated group significantly decreased blood glucose levels.
중성지방 분석Triglyceride Analysis
감태 추출물에 의한 체지방 감소효과를 가지는지 살펴보기 위해 10주간 시험물질 투여 후 채혈을 실시하고, 8,000 rpm에서 20 분간 원심 분리하여 혈청을 분리한 후, 냉동보관 하였다(-18). 혈중 중성지방은 kit(아산제약, AM 157S-K)을 이용하여 측정하였으며, 효소시액 3 ml을 기준으로 표준액을 이용하여 정량선을 작성한 후, 냉동 보관된 혈청 중 20ul을 취하여 37 incubator에 10분간 방치한 후, 분광광도계에서 550 nm에서 측정하였다.In order to examine the effect of Ecklonia cava extract on body fat, blood samples were taken after administration of test substance for 10 weeks, and serum was separated by centrifugation at 8,000 rpm for 20 minutes, and then stored frozen (-18). Blood triglycerides were measured using a kit (Asan Pharmaceutical Co., AM 157S-K), and after the determination line was prepared using the standard solution based on 3 ml of enzyme solution, 20ul of frozen serum was taken for 10 minutes in a 37 incubator. After standing, it was measured at 550 nm in a spectrophotometer.
실험결과, 도 11에 나타낸 바와 같이 정상식이군(ND)과 비교하여 고지방식이군(HD)에서 유의적으로 혈중 중성지방 함량이 증가하였고, 양성대조군으로 사용된 가르시니아군(GAR 25), 감태 열수추출물 150mg (60H 150; 제조예 8)군과 감태 효소처리군 (R+R 150; 제조예 4)군에서 농도의존적으로 중성지방 함량이 감소하였다.As shown in FIG. 11, the blood triglyceride content was significantly increased in the high fat diet group (HD) as compared to the normal diet group (ND), and the garcinia group (GAR 25) used as a positive control group, Ecklonia hot-water extract In the 150 mg (60H 150; Preparation Example 8) group and Eckloniasis enzyme treatment group (R + R 150; Preparation Example 4) group, the triglyceride content was reduced in a concentration-dependent manner.
콜레스테롤 분석Cholesterol analysis
감태 추출물에 의한 혈중 총콜레스테롤에 미치는 영향을 평가하기 위하여 부검 시 분리한 혈청을 이용하였으며, 혈청 중 총콜레스테롤 성분 정량 검사용 kit(아산제약,AM 202-K)을 이용하여 측정하였다. 효소시액 3 ml을 기준으로 표준액을 이용하여 정량선을 작성한 후, 냉동 보관된 혈청 중 20ul을 취하여 37 incubator에 5분간 방치한 후, 분광광도계에서 500nm에서 측정하였다.In order to evaluate the effect of Ecklonia cava extract on blood total cholesterol, serum was isolated at autopsy and measured using a kit for quantitative testing of total cholesterol in serum (Asan Pharmaceutical Co., AM 202-K). After the standard line was prepared using the standard solution based on 3 ml of enzyme solution, 20ul of frozen serum was collected and left in a 37 incubator for 5 minutes, and measured at 500 nm with a spectrophotometer.
실험결과, 도 12에 나타낸 바와 같이 정상식이군(ND)과 비교하여 고지방식이군(HD)에서 총 콜레스테롤의 함량이 유의적으로 증가하였고, 본 발명 감태 열수추출물 처리군(제조예 8)에서 총 콜레스테롤 함량은 감소하는 것으로 나타났다. . As a result, as shown in FIG. 12, the total cholesterol content was significantly increased in the high fat diet group (HD) compared to the normal diet group (ND), and the total cholesterol in the Ecklonia cava hot-water extract treatment group (Preparation Example 8) of the present invention. The content was found to decrease. .
또, HDL-콜레스테롤의 경우, 도 13에 나타낸 바와 같이 본 발명 감태 추출물 150mg 식이군(제조예 8)과 본 발명 효소처리 150mg 식이군(제조예 4)에서 증가하였고, 본 발명 감태 추출물(제조예 4, 제조예 8)을 각각 25mg 처리한 군에서도 총 콜레스테롤 대비 HDL-콜레스테롤이 증가하였으므로 본 발명 감태 추출물 25mg 식이군에서도 체지방 감소효과를 가져올 것으로 사료된다.In addition, in the case of HDL-cholesterol, the Ecklonia cava extract 150mg of the present invention (Preparation Example 8) and the enzyme treatment 150mg dietary group of the present invention (Preparation Example 4) increased as shown in Figure 13, the present invention Ecklonia cava extract (Preparation Example) 4, Preparation Example 8) In the group treated with 25mg respectively, HDL-cholesterol was increased compared to the total cholesterol, so it is believed that the Ecklonia cava extract of the present invention will have a body fat reduction effect.
GOT/GPT 분석GOT / GPT Analysis
감태 추출물 투여에 의한 간 손상 정도를 측정하기 위해 대표적인 간 지표인 혈중 GOT/GPT의 농도를 측정하였다. In order to measure the degree of liver damage by administration of Ecklonia cava extract, the concentration of GOT / GPT in blood, a representative liver indicator, was measured.
GOT/GPT의 측정은 kit(아산제약,AM 101-K)을 Ritman-Frankel법을 이용하여 측정하였는데 기질액 1 ml을 기준으로 표준액을 이용하여 정량선을 작성한 후, 냉동 보관된 혈청 중 200ul을 취하여 37 incubator에서 GOT는 60분, GPT는 30분 각각 반응시켰다. 정색시액 1ml과 혼합하여 실온에 20분 방치, 0.4N 수산화나트륨 용액 10 ml을 잘 혼합하여 실온에서 10분간 반응 후, 분광광도계에서 505 nm에서 증류수를 대조군으로 하여 측정하였다. GOT / GPT was measured using Ritman-Frankel method for kit (Asan Pharmaceutical Co., AM 101-K), and 200ul of serum stored in frozen serum was prepared by using a standard solution based on 1 ml of substrate solution. GOT was reacted for 60 minutes and GPT for 30 minutes in 37 incubators. After mixing with 1 ml of color solution, the mixture was allowed to stand at room temperature for 20 minutes, and 10 ml of 0.4N sodium hydroxide solution was mixed well for 10 minutes at room temperature, and distilled water was measured as a control at 505 nm on a spectrophotometer.
GOT/GPT는 간이 손상되었을 때 혈중에 높은 함량으로 존재하는 효소로서 혈청 중의 GOT/GPT 활성의 증가는 간 손상을 나타낸다.GOT / GPT is an enzyme that is present in blood at high levels when the liver is damaged. An increase in GOT / GPT activity in serum indicates liver damage.
실험결과, 도 14 내지 15에 나타낸 바와 같이 정상식이군과 비교했을 때 고지방식이군에서 상기 효소들의 활성이 2배 이상 증가하였으며, 가르시니아군 및 본 발명 감태 열수추출물 식이군(제조예 8), 본 발명 감태 효소처리 식이군(제조예 4)에서 상기 효소들의 활성이 감소하였다. As a result, as shown in Figure 14 to 15, the activity of the enzymes in the high-fat diet group increased more than two times as compared to the normal diet group, Garcinia group and the Ecklonia cava hot water extract diet group (Preparation Example 8), the present invention In the Ecklonia cava enzymatic group (Preparation Example 4), the activity of the enzymes was reduced.
인슐린 농도 측정Insulin concentration measurement
인슐린 농도 측정은 부검 시 분리한 혈청을 이용하여 마우스 인슐린 ELISA kit(catalog NO 80-INSMS-Eol)을 이용하여 혈청 인슐린 농도를 정량하였다. 표준액을 이용하여 정량선을 작성한 후, 냉동보관된 혈청 중 10ul를 취하여 working strength conjugate 75ul 씩 분주한 뒤, 실온에서 2시간(700-900 rpm) 방치하였다. 그 후 100ul 씩 TMB substrate를 분주하여 실온에서 15분 shaking 반응시키고 100ul stop solution을 취하여 분광광도계에서 450 nm에서 증류수를 대조군으로 하여 측정하였다. Insulin concentration was measured by using a mouse insulin ELISA kit (catalog NO 80-INSMS-Eol) was used to quantify the serum insulin concentration at the time of autopsy. After the determination line was prepared using the standard solution, 10ul of the frozen stored serum was dispensed by 75ul of each working strength conjugate, and then left at room temperature for 2 hours (700-900 rpm). Thereafter, 100 μl of TMB substrate was dispensed and shaken at room temperature for 15 minutes. A 100ul stop solution was taken to measure distilled water at 450 nm as a control in a spectrophotometer.
인슐린은 혈당을 조절하는 역할을 하며, 지방조직에서 비만 발병 위험성을 증가시키는 것으로 알려져 있다. 고지방식이 섭취 시 혈청 중의 인슐린 농도에 영향에 대하여 분석하였다. Insulin plays a role in regulating blood sugar and is known to increase the risk of obesity in adipose tissue. The effect of high-fat diet on serum insulin concentration was analyzed.
분석결과, 인슐린의 함량은 도 16에 나타낸 바와 같이 정상식이군(ND)과 비교하여 고지방식이군(HD)에서 유의적으로 인슐린의 함량이 증가하였고, 본 발명 감태 처리군 모두에서 인슐린의 혈중 함량이 유의적으로 감소하였다.As a result, the insulin content was significantly increased in the high fat diet group (HD) compared to the normal diet group (ND), as shown in Figure 16, the blood content of insulin in all the Ecklonia cava treatment group of the present invention Significantly decreased.
따라서, 본 발명 감태 추출물은 고지방식이로 인하여 증가한 인슐린의 함량을 낮추는 뛰어난 효과가 있음이 확인되었다.Therefore, the Ecklonia cava extract of the present invention was confirmed to have an excellent effect of lowering the increased insulin content due to high fat diet.
렙틴 함량분석Leptin Content Analysis
감태추출물이 지방세포의 분화의 마커로 사용되는 렙틴의 함량을 측정하기 위하여 부검 시 분리한 혈청을 이용하여 렙틴 ELISA kit(catalog NO ADI-900-019A)을 이용하여 혈청 렙틴 농도를 정량하였다. Assay buffer 100ul 기준에 표준액을 이용하여 정량선을 작성한 후, 냉동보관된 혈청 중 100ul를 취하여 실온에서 1시간 shaking 반응을 시켰다. Serum leptin concentrations were quantified using leptin ELISA kit (catalog NO ADI-900-019A) using serum isolated during autopsy to determine the leptin content used by Ecklonia cava extract as a marker of differentiation of adipocytes. After making the determination line using the standard solution to 100ul assay buffer, 100ul of frozen serum was taken and allowed to shake for 1 hour at room temperature.
이후, PBS 용액으로 washing 3번 반복한 후 100ul 씩 blue conjugate 분주하여 실온에서 30분간 방치했다. 이어서 washing 3번을 반복한 후, TMB substrate solution 100ul를 분주하여 실온에서 30분간 반응시킨 뒤, 100ul 씩 stop solution을 취하여 분광광도계 450nm에서 증류수를 대조군으로 측정하였다. Thereafter, washing with PBS solution was repeated three times, followed by dispensing blue conjugates at 100 ul for 30 minutes. After repeated washing 3 times, 100ul of TMB substrate solution was dispensed and reacted for 30 minutes at room temperature. Then, 100ul of stop solution was taken and distilled water was measured as a control in a spectrophotometer 450nm.
렙틴은 주로 지방세포에서 생산 및 분비되며 그 양은 체지방량에 비례하는 것으로 알려져 있다. Leptin is produced and secreted mainly from fat cells, and its amount is known to be proportional to body fat mass.
실험결과, 도 17에 나타낸 바와 같이 혈청 중의 렙틴 함량은 본 발명 감태 추출물 식이군에서 감소하였다. As a result, as shown in Figure 17, leptin content in serum was reduced in the Ecklonia cava extract diet group of the present invention.
실험예 8: 본 발명 감태 추출물처리에 따른 고지방식이 마우스의 간기능 분석Experimental Example 8: Liver function analysis of high fat diet mice according to the present invention Ecklonia cava extract treatment
간 조직 중의 중성지방 분석Triglyceride Analysis in Liver Tissue
감태 추출물에 의한 체지방 감소효과를 살펴보기 위해 10주간 시험물질을 투여 한 뒤, 간 조직을 척출하여 생리식염수로 씻어내고 수분을 여과지로 제거한 후 액체질소로 동결시켜 70에 보관하면서 간 조직 시료로 사용하였다. 획득한 간 조직을 세절하여 PBS buffer에 넣고 균질화 시킨 다음, 10,000, 4, 15 min 원심분리하여 상층액을 사용하였다. 간 조직의 중성지방은 kit(아산제약, AM 157S-K)을 이용하여 측정하였으며, 효소시액 3 ml을 기준으로 표준액을 이용하여 정량선을 작성한 후, 냉동 보관된 균질화시킨 간조직 20ul을 취하여 37 incubator에 10분간 방치한 후, 분광광도계 550 nm에서 측정하였다. To examine the effect of Ecklonia cava extract on body fat reduction, after administration of test substance for 10 weeks, hepatic tissue was extracted, washed with physiological saline solution, water was removed with filter paper, frozen with liquid nitrogen and stored at 70 to use as liver tissue sample. It was. The obtained liver tissue was cut and put into PBS buffer, homogenized, and centrifuged at 10,000, 4, and 15 min, and the supernatant was used. The triglycerides of liver tissue were measured using kit (Asan Pharmaceutical Co., AM 157S-K), and after making the determination line using the standard solution based on 3 ml of enzyme solution, take 20ul of homogenized liver tissue stored in frozen form 37 After standing in the incubator for 10 minutes, it was measured at 550 nm spectrophotometer.
본 발명에 따른 감태 추출물(제조예 4, 제조예 8)이 간 조직의 중성지방 함량에 미치는 영향을 분석하였다.The effect of Ecklonia cava extract (Preparation Example 4, Preparation Example 8) according to the present invention on the triglyceride content of liver tissue was analyzed.
실험결과, 도 18에 나타난 바와 같이 간의 중성지방 수치는 정상식이군(ND)과 비교하여 고지방식이군(HD)에서 유의적으로 증가하였고, 본 발명 감태 발효주정 추출물(제조예 8) 및 가수분해물(제조예 4) 식이군에서 유의적으로 중성지방 함량이 감소하였다. As a result, as shown in FIG. 18, triglyceride level of liver was significantly increased in the high fat diet group (HD) compared to the normal diet group (ND), and the present invention Ecklonia cava fermentation extract (Preparation Example 8) and hydrolyzate ( Preparation Example 4 The triglyceride content was significantly decreased in the diet group.
간 조직 중의 SOD 활성 분석SOD activity analysis in liver tissue
SOD는 동물의 거의 모든 조직에 분포되어 있으며, 그중 간 조직에 가장 많이 존재하는 것으로 알려져 있다. 이는 산화적 스트레스로부터 세포나 호기성 유기체를 보호하는 데 중요한 역할을 하는 효소이며, 이 효소의 작용기전은 효소에 있는 금속의 산화환원과 연관되어 superoxide radical의 전자를 전달하여 유독한 superoxide radical을 제거하는 것으로 알려져 있다. SOD is distributed in almost all tissues of animals, and is known to exist most in liver tissue. It is an enzyme that plays an important role in protecting cells and aerobic organisms from oxidative stress, and its mechanism of action is associated with the redox of metals in the enzymes to transfer electrons from superoxide radicals to remove toxic superoxide radicals. It is known.
간 조직 중의 SOD 활성분석은 kit(cayman, 706002)를 사용하였으며, 상기 실시예 4에 따라 수행한 실험동물의 간 조직을 각각 적출하여 SOD 추출용 buffer(20mM HEPES buffer, pH7.2, containing 1mM EGTA, 210mM mannitol, and 70mM sucrose)로 균질화 시킨 후 1,500, 4에서 5분 동안 원심분리하여 얻은 상층액을 효소원으로 사용하였다. 96-well plate에 sample과 SOD standard를 각각 10ul씩 분주하고 희석시킨 radical detector 200ul를 첨가한 다음 xantine oxidase 20ul를 넣었다. 실온에서 20분 동안 shaking 반응을 시킨 후, 분광광도계 450 nm에서 간 조직 중의 SOD활성을 측정하였다.SOD activity analysis in the liver tissue was used kit (cayman, 706002), the liver tissue of the experimental animal performed in accordance with Example 4 were extracted respectively SOD extraction buffer (20mM HEPES buffer, pH7.2, containing 1mM EGTA , 210mM mannitol, and 70mM sucrose),The supernatant obtained by centrifugation for 4 to 5 minutes was used as the enzyme source. 10ul of sample and SOD standard were dispensed into the 96-well plate, and 200ul of diluted radical detector was added, followed by adding 20ul of xantine oxidase. After shaking for 20 minutes at room temperature, Spectrophotometer at 450 nm SOD activity in liver tissue was measured.
실험결과, 도 19에 나타낸 바와 같이 간 조직 중의 SOD 활성은 고지방식이군에서 높게 나타났으며 본 발명에 제조예 4에 따라 제조된 감태 효소 추출물 처리군에서 SOD 활성이 유의적으로 감소하였다. As a result, as shown in FIG. 19, SOD activity in liver tissue was high in the high fat diet group, and SOD activity was significantly decreased in the Ecklonia cava extract treatment group prepared according to Preparation Example 4 of the present invention.
간 조직 중의 CAT 활성 분석CAT activity analysis in liver tissue
SOD 항산화효소는 superoxide radical을 과산화수소로 전환시키고 다시 이를 무독화시키는 GSH나 CAT를 기질로 이용하는 GPx에 의해 과산화수소를 무해한 물과 산소로 전환시켜 대사과정 중에 생성된 활성산소를 소거하는 역할을 하는 것으로 알려져 있다. SOD antioxidants are known to play a role in scavenging free radicals generated during metabolism by converting hydrogen peroxide into harmless water and oxygen by GPx, which converts superoxide radicals to hydrogen peroxide and then detoxifies them. .
간 조직 중의 CAT 활성분석은 kit(cayman, 707002)를 사용하였으며, 상기 실시예 4에 따라 수행한 실험동물의 간 조직을 각각 적출하여 CAT 추출용 buffer(50mM potassium phosphate,pH 7.0, containing 1mM EDTA)에 균질화 시켰으며, 10,000, 4에서 15분 동안 원심분리한 다음 상층액을 회수하여 사용하였다. 측정방법은 상층액, Formaldehyde standard, Positive control을 각각 20ul씩 분주하고, 1X assay buffer 100ul, MeOH 30ul를 첨가 후 hydrogen peroxide 20ul를 넣고 실온에서 20분 동안 방치하였다. 이후, Potassium hydroxide 30ul를 분주한 다음, purpald 30ul 첨가하여 실온에서 10분 방치한 뒤, Potassium periodate 10ul를 첨가하고 실온에서 5분 반응시킨 다음 분광광도계 540 nm에서 간 조직 중의 간손상에 관여하는 것으로 알려져 있는 CAT 활성을 측정하였다. CAT activity analysis in liver tissue was performed using kit (cayman, 707002). Liver tissues of the experimental animals carried out according to Example 4 were extracted, respectively, for CAT extraction buffer (50 mM potassium phosphate, pH 7.0, containing 1 mM EDTA).)It was homogenized to, and centrifuged for 15 minutes at 10,000, 4, and then the supernatant was collected and used. In the measurement method, 20ul of the supernatant, Formaldehyde standard, and positive control were each dispensed, and 100ul of 1X assay buffer and 30ul of MeOH were added, and 20ul of hydrogen peroxide was added thereto and left at room temperature for 20 minutes. After dissolving 30ul of Potassium hydroxide, and adding 30ul of purpald and leaving it for 10 minutes at room temperature, 10ul of Potassium periodate was added and reacted for 5 minutes at room temperature, and was known to be involved in liver damage in liver tissue at 540 nm. CAT activity was measured.
실험결과, 도 20에 나타낸 바와 같이 간 조직 중의 CAT 효소활성은 정상 식이군과 비교했을 때 고지방식이군에서 감소하였고, 본 발명에 제조예 4 및 제조예 8에 따라 제조된 감태 효소 추출물 처리군에서 CAT 효소활성이 정상수준으로 회복되었다. As a result, as shown in Figure 20 CAT enzyme activity in liver tissue was reduced in the high-fat diet group compared to the normal diet group, in the Ecklonia cava enzyme extract treatment group prepared according to Preparation Examples 4 and 8 in the present invention CAT enzyme activity was restored to normal levels.
따라서, 본 발명 감태 추출물이 고지방식이로 감소된 CAT 효소활성을 증가시켜 간기능을 회복시키는 것으로 사료된다. Therefore, the Ecklonia cava extract of the present invention is believed to restore liver function by increasing the reduced CAT enzyme activity in a high fat diet.
실험예 9: 본 발명 감태 추출물처리에 따른 고지방식이 마우스의 단백질 발현분석Experimental Example 9: Analysis of protein expression of high fat diet mice according to the present invention Ecklonia cava extract treatment
상기 실시예 4에 따라 수행한 실험동물의 간 조직을 각각 적출하여 비만 관련 인자의 단백질 발현 수준을 western blotting 법으로 분석하였다.  Liver tissues of the experimental animals performed according to Example 4 were extracted, and the protein expression levels of the obesity related factors were analyzed by western blotting.
단백질발현분석 결과, 도 21에 나타낸 바와 같이 지방 합성에 관여하는 것으로 알려져 있는 ACC-1, FAS 유전자는 고지방식이군에 비해 감태 처리 식이군에서 감소하였으나, 지방분해에 관여하는 것으로 알려져 있는 PPAR-, CD-36 유전자는 고지방식이군에 비해 감태 처리 식이군에서 증가하는 경향을 보여주었으며, 이는 효소 처리군에서 좀더 증가하였다. As a result of protein expression analysis, as shown in FIG. 21, ACC-1 and FAS genes, which are known to be involved in fat synthesis, were reduced in the Ecklonia cava diet group compared to the high fat diet group, but PPAR-, which is known to be involved in lipolysis, The CD-36 gene showed a tendency to increase in the ECO diet compared to the high-fat diet, which was increased in the enzyme treatment group.
비만 할수록 증가되는 OB-receptor의 발현이 고지방식이군에 비해 감태 처리군에서 감소하는 경향을 보여주었으며, acrp30 (adiponectin)은 간과 근육의 AMP Kinase를 활성화시켜 ACC-1의 발현을 억제하고 지방산 산화를 촉진시키는 역할을 하는 것으로 알려져 있다. 위의 ACC-1의 결과와 반대로 감태 처리군에서 증가하는 경향을 보여주어 지방산 산화를 촉진시키는 것으로 사료된다. The expression of OB-receptor increased with obesity tended to decrease in EAC group compared to high fat diet group.Acrp30 (adiponectin) activates AMP Kinase in liver and muscle to inhibit ACC-1 expression and reduce fatty acid oxidation. It is known to play a role in promoting. Contrary to the results of the above ACC-1, it was found to increase in the Ecklonia cava group, thereby promoting fatty acid oxidation.
장 및 증식, 분화에 관여하는 것으로 알려져 있는 ERK, AKT, NF-B 유전자의 발현수준을 살펴보았다. The expression levels of ERK, AKT, and NF-B genes, which are known to be involved in intestinal proliferation and differentiation, were examined.
실험결과, 고지방식이군에 비해 가르시니아, 감태처리군에서 ERK와 NF-B의 발현 수준을 감소시켜 증식을 억제하는 것으로 나타났다. Experimental results showed that the Garcinia and Ecklonia cava treatments suppressed proliferation by decreasing the expression levels of ERK and NF-B in the high fat diet group.
따라서, 고지방식이에 본 발명 감태 열수추출물 및 효소 처리 추출물의 첨가는 지방대사 관련 단백질의 발현수준을 조절하고, 인슐린 함량을 낮추어 혈당을 조절할 뿐만 아니라, 중성지방과 같은 콜레스테롤 대사에도 영향을 미쳐 비만과 같은 지질대사 이상을 개선시키는 데 긍정적인 영향을 나타내어 항비만용 소재로 이용가능하다고 사료된다. Therefore, the addition of the present invention Ecklonia cava hot water extract and enzyme-treated extract to a high-fat diet regulates the expression level of fat metabolism-related protein, lowers insulin content to regulate blood sugar, and also affects cholesterol metabolism such as triglycerides, obesity It can be used as an anti-obesity material because it has a positive effect on improving lipid metabolism abnormalities.
이상 설명한 바와 같이, 본 발명은 체중감소 효과와 항비만 효과를 갖는 감태의 효소 가수분해물, 발효주정 추출물 및 열수 추출물을 제공하는 뛰어난 효과가 있으므로 건강기능성식품산업 및 생물의약산업상 매우 유용한 발명이다.As described above, the present invention has an excellent effect of providing enzyme hydrolyzate, fermented alcohol extract and hot water extract of Ecklonia cava having a weight loss effect and anti-obesity effect, and thus is a very useful invention for the health functional food industry and the biopharmaceutical industry.

Claims (7)

  1. 감태에 프로텍스 6엘, 라피다제 프레스 엘, 로하멘트 씨엘 효소로 이루어진 군으로부터 선택되는 1종 이상의 가수분해 효소를 첨가하여 수득한 것이 특징인 체지방 감소 활성을 갖는 감태 가수분해물. Ecklonia cava hydrolyzate having body fat reducing activity, characterized in that obtained by adding at least one hydrolase selected from the group consisting of Protex 6L, Lapidase press L, Lohament CI enzyme.
  2. 제 1항 기재의 감태 가수분해물을 함유하는 것을 특징으로 하는 체중 감소 기능성 건강식품 조성물.A weight loss functional health food composition, comprising the E. coli hydrolyzate of claim 1.
  3. 제 1항 기재의 감태 가수분해물을 유효성분으로 함유하는 것을 특징으로 하는 비만 예방 및 치료용 약학적 조성물.A pharmaceutical composition for the prevention and treatment of obesity, comprising the E. coli hydrolyzate of claim 1 as an active ingredient.
  4. 감태를 추출용매에 침지하여 수득한 것이 특징인 체지방 감소활성을 갖는 감태 추출물.Ecklonia cava extract having body fat reducing activity, characterized in that obtained by immersing Ecklonia cava in the extraction solvent.
  5. 제 4항에 있어서, 상기 추출용매는 물 또는 물과 알코올류 혼합물로 추출하여 수득된 것이 특징인 체지방 감소활성을 갖는 감태 추출물.5. The Ecklonia cava extract according to claim 4, wherein the extractant is obtained by extraction with water or a mixture of water and alcohols.
  6. 제 4항 또는 제 5항 기재의 감태 추출물을 함유하는 것을 특징으로 하는 체중 감소 기능성 건강식품 조성물.A weight loss functional health food composition comprising the Ecklonia cava extract according to claim 4.
  7. 제 4항 또는 제 5항 기재의 감태 추출물을 함유하는 것을 특징으로 하는 비만 예방 및 치료용 약학적 조성물.A pharmaceutical composition for the prevention and treatment of obesity, comprising the extract of Ecklonia cava according to claim 4 or 5.
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