WO2014189176A1 - Extrait d'ecklonia cava permettant la perte de poids et son procédé de préparation - Google Patents

Extrait d'ecklonia cava permettant la perte de poids et son procédé de préparation 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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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

La présente invention présente un effet excellent de fourniture d'un hydrolysat d'enzyme, d'un extrait alcoolique fermenté et d'un extrait à l'eau chaude d'Ecklonia cava qui présente des effets amincissants et anti-obésité.
PCT/KR2013/007798 2013-05-20 2013-08-30 Extrait d'ecklonia cava permettant la perte de poids et son procédé de préparation WO2014189176A1 (fr)

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KR101976839B1 (ko) * 2017-03-17 2019-05-09 한남대학교 산학협력단 해조류 추출물 및 로즈마리 추출물을 유효성분으로 포함하는 비만 예방 또는 치료용 약학 조성물
WO2020130208A1 (fr) * 2018-12-21 2020-06-25 아쿠아그린텍(주) Composition de régulation alimentaire comprenant un extrait d'ecklonia cava et procédé de préparation associé
KR102400107B1 (ko) * 2022-01-20 2022-05-19 주식회사 비상썬라이즈 수면 유도 및 다이어트용 건강기능식품 조성물 및 이의 제조 방법
KR102642516B1 (ko) 2022-12-28 2024-03-05 (주)에스앤디 감태추출물혼합물을 유효성분으로 포함하는 호흡기 질환 예방, 개선 또는 치료용 조성물 및 제조방법

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