WO2018066969A1 - Composition pharmaceutique comprenant de l'arazyme en tant que principe actif pour la prévention ou le traitement d'une maladie métabolique - Google Patents

Composition pharmaceutique comprenant de l'arazyme en tant que principe actif pour la prévention ou le traitement d'une maladie métabolique Download PDF

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WO2018066969A1
WO2018066969A1 PCT/KR2017/011095 KR2017011095W WO2018066969A1 WO 2018066969 A1 WO2018066969 A1 WO 2018066969A1 KR 2017011095 W KR2017011095 W KR 2017011095W WO 2018066969 A1 WO2018066969 A1 WO 2018066969A1
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arazyme
metabolic diseases
pharmaceutical composition
prevention
treatment
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PCT/KR2017/011095
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Korean (ko)
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박호용
정태숙
신동하
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주식회사 인섹트 바이오텍
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/4886—Metalloendopeptidases (3.4.24), e.g. collagenase
    • 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
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/328—Foods, ingredients or supplements having a functional effect on health having effect on glycaemic control and diabetes

Definitions

  • the present invention relates to a pharmaceutical composition for preventing or treating metabolic diseases containing arazyme as an active ingredient.
  • Diabetes mellitus is a metabolic disease, such as insufficient insulin secretion or normal functioning. Diabetes mellitus is characterized by high blood sugar, which increases the concentration of glucose in the blood. And release glucose into the urine.
  • Diabetes is one of the most important adult diseases in the world. In Korea, the prevalence of diabetes reaches 10%, and the number of diabetics is over 240 million worldwide, and increased to 380 million worldwide by 2025. Of these, 60 percent will occur in Asia, according to the 2009 American Medical Association (JAMA).
  • the main cause of type 2 diabetes which accounts for 90% of the diabetic patients in Korea, is the dietary habits of high calorie, high fat and high protein according to the westernization of diet.
  • the prevalence of diabetes continues to increase due to fast food intake and lack of exercise.
  • Diabetes mellitus in Korea is constitutionally 'dry obesity', and most of the patients have a normal range of body weight, but most of them have abdominal obesity.
  • three out of four diabetics are overweight or obese.
  • glycemic control is mainly achieved by insulin secreted from the pancreas.
  • the abdominal obesity rate of diabetics in Korea is about 56% for women and 41% for men (2016, Korea Centers for Disease Control and Prevention). They may eat insulin or reduce their activity.
  • Koreans Compared with westerners, Koreans have lower insulin secretion ability, have higher complications such as hypertension, hyperlipidemia, and diabetic nephropathy, and have low compliance with medications. There is a need for a comprehensive screening project to reduce the incidence of complications for early detection of diabetes and the risk of diabetes.
  • Fatty liver refers to a condition in which abnormal fat is accumulated in liver cells, and a medical condition refers to a pathological condition in which the neutral lipid content exceeds 5% of the total liver weight.
  • fatty liver is characterized by alcoholic fatty liver disease (ALD) caused by persistent and excessive drinking and nonalcoholic fatty liver disease (NAFLD), which has little alcohol intake but similar liver tissue findings to alcoholic fatty liver. It can be divided into two.
  • ALD alcoholic fatty liver disease
  • NAFLD nonalcoholic fatty liver disease
  • non-alcoholic fatty liver disease can be caused by the causes of obesity, diabetes, hyperlipidemia, drugs, etc., regardless of drinking Non-alcoholic steatohepatitis (NASH), advanced fibrosis and cirrhosis, which show simple steatosis and hepatocellular inflammation that do not accompany the inflammatory response over time. It means a wide range of diseases up to and including.
  • NASH Non-alcoholic steatohepatitis
  • advanced fibrosis and cirrhosis which show simple steatosis and hepatocellular inflammation that do not accompany the inflammatory response over time. It means a wide range of diseases up to and including.
  • insulin resistance drugs metalformin, pioglitazone, rosiglitazone
  • hyperlipidemia drugs that treat and improve fatty liver by correcting risk factors.
  • therapeutic agents clofibrate, gemfibrozil, bezafibrate, atorvastatin, simvastatin
  • hepatoprotective agents ursodeoxycholic acid and taurine
  • antioxidants vitamine E antioxidants vitamine E
  • nutritional supporters lectin, betaine, N-acetylcystein
  • the existing therapeutic agents are drugs used as symptomatic agents rather than essential therapeutic agents in terms of efficacy, they cannot be seen as target effects.
  • the drug approved by the Ministry of Food and Drug Safety for the efficacy of non-alcoholic fatty liver treatment is currently only one item in Korea, and the market size of this drug has been decreasing since 2008, which indicates that the effect of the drug is insufficient. It is believed to be due to a fundamental problem. Therefore, until now, there is almost no pharmacological agent capable of treating nonalcoholic fatty liver, and thus there is an urgent need to develop an appropriate therapeutic agent having no side effects and excellent safety even for long-term administration.
  • arazyme derived from the proteolyticus strain was administered to a nonalcoholic fatty liver animal model, it was confirmed that plasma glycated hemoglobin (HbA1c), total cholesterol, triglycerides and non-esterified fatty acids (NEFA) were reduced.
  • HbA1c plasma glycated hemoglobin
  • NEFA non-esterified fatty acids
  • An object of the present invention to provide a pharmaceutical composition for the prevention or treatment of metabolic diseases containing arazyme (Arazyme) as an active ingredient.
  • the present invention provides a pharmaceutical composition for the prevention or treatment of metabolic diseases containing arazyme (Arazyme) as an active ingredient.
  • the present invention also provides a health functional food for improving metabolic diseases containing arazyme (Arazyme) as an active ingredient.
  • the present invention also provides a method for preventing or treating metabolic disease, comprising administering to a subject a pharmaceutically effective amount of arazyme.
  • the present invention also provides arazyme (Arazyme) for use as a composition for preventing or treating metabolic diseases.
  • the present invention provides arazyme (Arazyme) for use as a dietary supplement for preventing or improving metabolic diseases.
  • Aranicolaa proteoritis of the present invention Aranicola Arazyme produced by proteolyticus reduces plasma glycated hemoglobin (HbA1c), total cholesterol, triglycerides and non-esterified fatty acids (NEFA) in animal models and improves glucose-loaded glucose tolerance in high-fat dietary animal models In addition, it promotes insulin secretion and synthesis in pancreatic beta cells and at the same time improves insulin resistance in hepatocytes and inhibits lipid accumulation and liver fibrosis, and thus may be useful as a pharmaceutical composition for preventing or treating metabolic diseases.
  • HbA1c plasma glycated hemoglobin
  • NEFA non-esterified fatty acids
  • FIG. 1 is a diagram showing the expression changes of PCSK1 / 3 and PCSK2 genes that regulate the synthesis of Ins1 and Ins2 insulin genes and insulin proteins in palmitic acid (PA) -induced MIN6 pancreatic beta cells.
  • PA palmitic acid
  • Figure 2 is a diagram showing the expression change of phosphorylated AKT protein reduced by palmitic acid in hepG2 cells, hepatocytes.
  • Figure 3 is a diagram showing the degree of fibrosis in liver tissue through sirius red staining in the liver of a high-fat diet-induced nonalcoholic fatty liver mouse model.
  • Figure 4 is a diagram showing the change in the size of the pancreatic islet (pancreatic islet) in the pancreatic tissue of the high fat diet-induced non-alcoholic fatty liver mouse model.
  • the present invention provides a pharmaceutical composition for the prevention or treatment of metabolic diseases containing arazyme (Arazyme) as an active ingredient.
  • the arazyme is preferably a polypeptide consisting of an amino acid sequence set forth in SEQ ID NO: 1, Aranicola proteoritis ( Aranicola proteolyticus ) strain may be preferred, but is not limited thereto.
  • the metabolic disease is preferably any one selected from the group consisting of type 2 diabetes, dyslipidemia, insulin resistance, and nonalcoholic fatty liver, but is not limited thereto.
  • the arazyme preferably increases the expression of the insulin gene or increases the secretion of insulin, and the insulin gene is preferably Ins1 or Ins2, but is not limited thereto.
  • the arazyme preferably increases the expression of PCSK1 / 3 or PCSK2 gene and phosphorylation of AKT protein, but is not limited thereto.
  • the metabolic disease is most preferably type 2 diabetes with nonalcoholic fatty liver disease (NAFLD), but is not limited thereto.
  • NAFLD nonalcoholic fatty liver disease
  • the arazyme reduces the levels of AST and ALT, which are indicators of hepatotoxicity in the blood, but is not limited thereto.
  • the arazyme preferably inhibits fibrosis and an increase in the size of pancreatic islets. Do not.
  • Arazyme of the present invention comprises the steps of 1) obtaining a culture by culturing the aranicola proteoricicus strain; 2) filtering the culture solution to obtain a supernatant; And 3) purifying the arazyme contained in the supernatant using a resin (WO 01/57222).
  • Aranicola proteolyticus strain as a microorganism to produce arazyme, and Aranicola proteorium of Accession No. KCTC 0268BP, deposited on July 29, 1996 with the Korea Biotechnology Research Institute Gene Bank. It is more preferable to use Cous HY-3, but is not limited thereto.
  • Aranicola Proteoricicus HY-3 strain is an aerobic Gram-negative bacterium isolated from the intestine of the spider's intestine, and has a spherical and motility of 0.5 to 0.8 mm in size, positive for catalase, and negative for oxidase. Reaction (Korean Patent No. 0220091). In the present invention, arazyme obtained by the above method was used.
  • arazyme obtained by the above method it is preferable to use arazyme obtained by the above method, and more preferably, but not limited to using arazyme produced by the following method.
  • the raw material used for the cultivation of the Aranicola proteoricicus strain is preferably at the level of medicines that are more comfortable than pure purified products and capable of producing high-purity results.
  • ammonium sulfate precipitation or acetone precipitation
  • arazyme is recovered by centrifugation and filtration. This is because most other proteins produced by microorganisms use different arazyme and precipitation concentrations.
  • a high-concentration arazyme in the form of a solution obtained by purification of primary impurities using membrane filtration and finally pure separation and purification using an ultrafilter is used in powder form through a freeze dryer.
  • Arazyme obtained by the above production method may be included as long as the protein is substantially purified according to any purification method of the protein.
  • the purified protein may be appropriately selected or combined with column chromatography, filters, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric point electrophoresis, dialysis or recrystallization. It is preferred that the protein is substantially purified, but is not limited thereto.
  • arazyme encoded from the DNA may be obtained using a protein expression system well known to those skilled in the art, and may be recovered and purified from a culture of cells expressing arazyme.
  • aranicola proteoritis HY-3 strain for the production of arazyme can be produced in a variety of industrial media, and the culture broth produced can also be isolated and purified by various methods.
  • lipid content such as triglyceride (TG) and total cholesterol (TC) is decreased in liver tissue of the animal model ingested arazyme (Table 5), and the degree of fibrosis in liver tissue is suppressed.
  • FIG. 3 it was confirmed that an increase in pancreatic islet size in the pancreatic tissue was suppressed.
  • the pharmaceutical composition containing arazyme of the present invention can be usefully used for the prevention and treatment of metabolic diseases.
  • arazyme of the present invention may contain one or more active ingredients exhibiting the same or similar functions.
  • it may be prepared further comprising one or more pharmaceutically acceptable carriers.
  • Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components, as necessary, including antioxidants, buffers, Other conventional additives such as bacteriostatic agents can be added.
  • diluents, dispersants, surfactants, binders and lubricants may be additionally added to formulate main formulations, powders, tablets, capsules, pills, granules or injections, such as aqueous solutions, suspensions, emulsions and the like.
  • it may be preferably formulated according to each disease or component by a suitable method in the art or using a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).
  • compositions for the prophylaxis and treatment of metabolic diseases of the present invention can be administered parenterally (eg, intravenously, subcutaneously, intraperitoneally or topically) or orally, depending on the desired method.
  • parenterally eg, intravenously, subcutaneously, intraperitoneally or topically
  • the range varies depending on body weight, age, sex, health condition, diet, administration time, administration method, excretion rate and severity of disease.
  • the daily dose of arazyme according to the present invention is 0.01 to 5000 mg / kg, preferably 0.01 to 10 mg / kg, and more preferably administered once to several times a day.
  • the present invention also provides a method for preventing or treating metabolic disease, comprising administering to a subject a pharmaceutically effective amount of arazyme.
  • the present invention also provides arazyme (Arazyme) for use as a composition for preventing or treating metabolic diseases.
  • the present invention also provides arazyme (Arazyme) for use as a dietary supplement for preventing or improving metabolic diseases.
  • the arazyme is preferably a polypeptide consisting of an amino acid sequence set forth in SEQ ID NO: 1.
  • the present invention provides a dietary supplement for improving metabolic diseases containing arazyme as an active ingredient.
  • the metabolic disease is preferably type 2 diabetes with nonalcoholic fatty liver disease (NAFLD), but is not limited thereto.
  • NAFLD nonalcoholic fatty liver disease
  • the arazyme of the present invention When the arazyme of the present invention is used as a food additive, the arazyme may be added as it is or may be used together with other foods or food ingredients, and may be appropriately used according to a conventional method. Arazyme is obtained and used in the same manner as described above.
  • the mixed amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment).
  • the arazyme of the present invention is added in an amount of 0.01 to 10 parts by weight, preferably 0.05 to 1 part by weight based on the raw materials.
  • the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety. .
  • Examples of the food to which the substance may be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, dairy products including other noodles, gum, ice cream, various soups, beverages, tea, drink, Alcoholic beverages and vitamin complexes, etc., includes all the health functional foods in the conventional sense.
  • the health beverage composition of the present invention may contain various flavors or natural carbohydrates, etc. as additional components, as in the general beverage.
  • Natural carbohydrates described above are glucose, monosaccharides such as fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin, cyclodextrin, sugar alcohols such as xylitol, sorbitol, and erythritol.
  • sweetener natural sweeteners such as tautin and stevia extract, synthetic sweeteners such as saccharin, aspartame, and the like can be used.
  • the proportion of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the beverage composition of the present invention.
  • arazyme contains various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols, carbonated drinks It can be added to a carbonation agent etc. used for.
  • Arazyme may also be added to the flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components can be used independently or in combination. The proportion of such additives is not critical but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of arazyme of the present invention.
  • Aranicola proteoricicus HY-3 strain (KCTC 0268BP) was cultured in a medium (bacto tryptone 0.5%, yeast extract 0.5%, sodium chloride 0.1%, 0.05% potassium chloride, 0.02% calcium chloride and 0.02% magnesium sulfate) were incubated at 22 ° C. for 18 hours.
  • the culture solution was separated from the cells and the supernatant using 2 ⁇ m membrane filtration, and the separated supernatant was concentrated using membrane filtration of 10 kDa.
  • the arazyme of the present invention basically has anion properties
  • the ion exchange resin and the ion exchange resin using DEAE-cellulose which have been pretreated with 50 mM tris-hydrochloric acid buffer (pH 7.6), and 20 mM Purification was carried out using a gel filtration exchange resin using Sephadex G-75 pretreated with Tris-HCl buffer (pH 7.6).
  • the purified enzyme solution was subjected to electrophoresis with 10% SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel) to confirm the band tendency.
  • SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel
  • MIN6 pancreatic beta cells a mouse pancreatic beta ( ⁇ ) cell line, were treated with 5% CO at 37 ° C. using DMEM (Hyclone) medium containing 15% FBS (Hyclone), 100 units / ml penicillin and 100 ⁇ g / ml streptomycin. 2 culture was incubated .
  • HepG2 cells a human hepatocellular carcinoma cell line, were treated with low concentration of glucose DMEM (5 mM, Hyclone) medium containing 10% FBS (Hyclone), 100 units / ml penicillin, and 100 ⁇ g / ml streptomycin. 2 culture was incubated .
  • glucose DMEM 5 mM, Hyclone
  • FBS FBS
  • mice Male animals were used as male C57BL / 6J mice (SLC, Japan). The pretreated animals were adapted to the laboratory environment with free feeding of AIN-76A diet and water for 3 weeks, and then male 7-week-old male mice in good health were used for the experiment. The experimental groups were classified as follows and 10 mice per group were used:
  • Test group supplemented with arazyme (0.025%, wt / wt diet) of the present invention to a high fat diet.
  • the experimental group was tested for 12 weeks to observe the lipid lowering, antidiabetic and hepatoprotective efficacy.
  • the experiment was carried out after approval of KRIBB-ACE-16050 from the Biosafety Ethics Committee of Korea Research Institute of Bioscience and Biotechnology.
  • the environment of the animal breeding room was maintained at constant temperature (25 ⁇ 2 °C), constant humidity (50 ⁇ 5%) and photoperiod of 12 hours (lighting 07:00 ⁇ 19:00), experimental animals 3 to 4
  • the animals were reared separately, and the diet and drinking water were taken freely. Dietary intake and body weight were measured and recorded at a regular time every week, and after fasting at 12-week intervals for 2 hours, blood was collected from the posterior eye and venous gun using capillary tubes to prevent coagulation using EDTA, and blood biochemical tests. For 30 minutes after blood collection, the plasma was separated by centrifugation at 3,000 rpm and 4 ° C. for 15 minutes, and stored at ⁇ 70 ° C. for analysis.
  • RNA stabilization solution Qiagen
  • results between the negative control group, the control group and the test group were expressed as mean ⁇ standard deviation.
  • the difference between the groups was one-way ANOVA followed by Turkey hoc test (JMP® software, SAS Institute, USA). Less than ( P ⁇ 0.05), statistical significance was determined.
  • the present inventors experimented to determine the effect of arazyme (arazyme) on insulin secretion ability in pancreatic beta cells.
  • pancreatic beta cells cultured in Example ⁇ 2-1> in a 24-well plate were dispensed with 1 ⁇ 10 5 cells per well, and a 5% CO 2 incubator at 37 ° C. Incubated at. After 48 hours, the cells were first replaced with glucose-free DMEM medium and left to stand for 60 minutes in cell hunger (fasting), and then the arazyme prepared in Example 1 prior to DMEM medium replacement. Were treated with 1 and 5 ⁇ g / ml, and reacted for 30 minutes, and then replaced with DMEM medium containing 30 mM glucose.
  • the control group was used without the addition of arazyme under the same conditions, and insulin secreted into the medium was measured using an ELISA insulin kit (Alpco diagnostics) (Martinez SC et al., Diabetes. 55: 1581-1591, 2006).
  • ELISA insulin kit Alpha diagnostics
  • the arazyme of the concentration was treated, it was confirmed whether or not to show the toxicity to the cells by concentration compared to the control group untreated group, the results of the experiment are shown in Table 1 below.
  • the insulin secretion of pancreatic beta cells induced by high glucose (30 mM) was 29.5% and 76.0% in the arazyme pretreated at 1 and 5 ⁇ g / ml concentrations, respectively. It was confirmed that the increase to (Table 1). It was also confirmed that arazyme had no toxicity to cells at these concentrations.
  • the present inventors experimented to determine the effect of arazyme (arazyme) on the expression of insulin-related genes in pancreatic beta cells (MIN6).
  • the expression level of the gene is expressed as the number of PCR cycles at the point where the cDNA is amplified and the fluorescence reaches saturation, which is corrected to the value for glycaldehyde 3-phosphate dehydrogenase (GAPDH) and finally the relative value to the control group. Calculated and calculated.
  • GPDH glycaldehyde 3-phosphate dehydrogenase
  • the present inventors experimented as follows to determine the effect of arazyme (arazyme) on lipid accumulation in hepatocytes (HepG2).
  • Example ⁇ 2-1> hepatocytes cultured in Example ⁇ 2-1> were placed in a 6-well plate (5-1 10 5 cells per well) and cultured in a 37% 5% CO 2 incubator. After 24 hours, 0.01, 0.05, 0.1 and 0.5 ⁇ g / ml of arazyme prepared in Example 1 were added to 0.2 mM of palmitic acid (PA), a triglyceride accumulation inducer, in each well. Each medium was replaced with the added medium and treated for 24 hours. As a control group, no sample was added under the same conditions, and the content of triacylglycerol (TG) accumulated by harvesting HepG2 cells was measured using a TG measurement kit (Asan Pharmaceutical). In addition, when the arazyme of the concentration was treated, it was also confirmed whether or not to show the toxicity to the cells by concentration compared to the control group untreated. The results of the experiment are shown in Table 3 below.
  • HepG2 cells were cultured for 24 hours, and then 0.2 mM of palmitic acid (PA) and concentration (0.01, 0.05, 0.1 and 0.5 ⁇ g / ml) in each well. Arazyme was treated and reacted for 24 hours. In the next step, 0.2 mM of insulin was treated for 20 minutes, and HepG2 cells were harvested and lysed to quantify the protein. 40 ⁇ g of protein was separated by SDS-PAGE, transferred to PVDF membrane, and then reacted with BSA-block (CANDOR bioscience, Germany) for 1 hour to block nonspecific proteins on PVDF membrane. After washing, the cells were reacted with primary AKT or phospho-AKT antibody for 24 hours.
  • PA palmitic acid
  • concentration 0.01, 0.05, 0.1 and 0.5 ⁇ g / ml
  • Example ⁇ 3-1> blood was collected from each experimental animal to separate plasma, and fasting glucose, fast fatty acid (nonesterified fatty acid (NEFA), and liver function). Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) concentrations were measured by biochemical automated analyzer (Hitachi-720, Hitachi Medical, Japan). In addition, glycated hemoglobin (HbA1c) was measured using an EASY A1C glycated hemoglobin cartridge (Inpopia, Korea). Insulin resistance index (HOMA-IR) was measured by using Insulin kit (ELISA kit, Alpco diagnostics). The concentration was calculated and measured. The concentrations of triglyceride (TG) and total cholesterol (TC), which are indicators of lipid content, were measured using a separate kit (Asan Pharmaceutical). The results of the experiment are shown in Table 4 below.
  • Plasma Lipid Biochemical Indicators Negative Control Control Test group Fasting Blood Sugar (mg / dL) 126.4 ⁇ 10.3 169.0 ⁇ 12.9 145.8 ⁇ 7.9 Glycated hemoglobin (%) 5.05 ⁇ 0.09 5.41 ⁇ 0.12 5.15 ⁇ 0.06 Insulin resistance index 6.88 ⁇ 1.30 19.12 ⁇ 4.73 16.39 ⁇ 3.03 Free fatty acid (mEq / L) 2.68 ⁇ 0.13a 2.73 ⁇ 0.11 2.55 ⁇ 0.16 Triglycerides (mg / dL) 118.2 ⁇ 7.5 142.7 ⁇ 9.5 118.4 ⁇ 4.2 Total Cholesterol (mg / dL) 144.5 ⁇ 6.4 183.1 ⁇ 9.4 170.6 ⁇ 7.2 AST (IU / L) 95.6 ⁇ 6.1 117.4 ⁇ 5.8 105.8 ⁇ 11.3 ALT (IU / L) 26.0 ⁇ 3.6 45.3 ⁇ 10.2 41.5 ⁇ 5.7
  • Triglycerides were significantly increased to 118.2 ⁇ 7.5 mg / dl in the negative control group and 142.7 ⁇ 9.5 mg / dl in the control group, while those in the arazyme group were significantly reduced to 118.4 ⁇ 4.2 mg / dl ( P ⁇ 0.05).
  • Total cholesterol was 144.5 ⁇ 6.4 mg / dl negative control group, the control group intake of arazyme compared to 183.1 ⁇ 9.4 mg / dl control group was confirmed to decrease to 170.6 ⁇ 7.2 mg / dl (Table 4).
  • the control group fed the high-fat diet showed a symptom of fatty liver, and the contents of AST and ALT, which are indicative of blood hepatotoxicity, were increased compared to the negative control group which received the basic diet for 12 weeks.
  • the AST content decreased to 95.8 ⁇ 6.1 IU / L in the negative control group and 117.4 ⁇ 5.8 IU / L in the control group, but decreased to 105.8 ⁇ 11.3 IU / L in the test group ingested with arazyme.
  • the negative control group was 26.0 ⁇ 3.6 IU / L and the control group was 45.3 ⁇ 10.2 IU / L, while the test group ingested arazyme decreased to 41.5 ⁇ 5.7 IU / L.
  • the protective effect was confirmed (Table 4).
  • the present inventors experimented as follows to determine the effect of arazyme in the nonalcoholic fatty liver animal model on the triglyceride (TG) and total cholesterol (TC) content in liver tissue.
  • TG triglyceryl
  • TC total cholesterol
  • the amount of triglyceride per weight (g) of liver was 6.2 ⁇ 0.5 mg in the negative control group and 4.7 ⁇ 0.2 mg in the test group using arazyme compared to 6.5 ⁇ 0.2 mg in the control group. It was confirmed that the amount of fat decreased by 27.7% ( P ⁇ 0.05).
  • the total cholesterol per liver weight was 63.5 ⁇ 4.5 mg in the negative control group and significantly increased to 90.4 ⁇ 8.2 mg in the control group, while the test group ingested arazyme was 67.6 ⁇ 2.6 mg, indicating a 25.2% reduction in total cholesterol. (Table 5) ( P ⁇ 0.05).
  • the present inventors conducted the following experiment to determine the effect on the degree of liver fibrosis when ingested arazyme in a non-alcoholic fatty liver animal model.
  • Liver tissues extracted from each experimental animal of the negative control group, the control group and the arazyme ingested Example ⁇ 3-1> were measured through the sirius red staining to measure the accumulation of collagen. The degree of fibrosis in liver tissue was compared by high fat diet.
  • the present inventors separated the pancreas of a non-alcoholic fatty liver animal model ingested with arazyme to determine whether the arazyme improved blood glucose, glycated hemoglobin, and insulin resistance indexes were related to pancreatic tissue function. Morphological analysis was carried out as follows.
  • Each mouse of the negative control group, the control group and the test group ingested arazyme of Experimental Example ⁇ 3-1> was fixed with 4% paraformaldehyde (paraformaldehyde) at room temperature for 1 hour after CO 2 treatment, and then the paraffin block was removed.
  • a standard H / E stain was performed.
  • the stained tissues were imaged using an Olympus BX61 (Olympus, Japan) equipped with a digital camera (Olympus DP71) and pancreatic islet using Metamorph imaging software (Molecular Devices, Sunnyvale, CA, USA). The size of was measured.
  • the size of the pancreatic islet is negative control (9.6 ⁇ 1.1) x 10 3 mm 2 and the control (24.7 ⁇ 3.2) x 10 3 mm 2 is the size of the pancreatic islet by the high-fat diet Was significantly increased, but in the test group ingested arazyme (18.6 ⁇ 1.4) x 10 3 mm 2 it was confirmed that the size of the pancreatic islet significantly reduced (Fig. 4). This is
  • the airtight cloth was filled to prepare a powder.
  • tablets were prepared by tableting according to a conventional method for producing tablets.
  • the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.
  • Arazyme was dissolved in a suitable volume of main sodium chloride BP, the pH of the resulting solution was adjusted to pH 7.6 with dilute hydrochloric acid BP, and the volume was adjusted with a main volume of sodium chloride BP and thoroughly mixed.
  • the solution was filled into a 5 ml Type I ampoule of clear glass, encapsulated under an upper grid of air by dissolving the glass, autoclaved at 120 to 15 minutes or more to prepare an injection solution.
  • the powders, tablets, capsules, pills, and granules of Formulation Example 1 may be applied to foods, and foods containing the culture solution of aranicola proteoritus or arazyme separated therefrom are prepared as follows. It was.
  • Foods for health promotion by adding 0.1 to 10.0 parts by weight of the culture solution of Aranicola proteoriticus or the arazyme separated therefrom to flour, and preparing breads, cakes, cookies, crackers and noodles using the mixture in a conventional manner.
  • Brown rice, barley, glutinous rice, and yulmu were alphad by a known method, and then dried and roasted to prepare a powder having a particle size of 60 mesh.
  • Black beans, black sesame seeds, and perilla were also steamed and dried by a known method, and then ground to a powder having a particle size of 60 mesh.
  • Araniacola proteoricicus medium or arazyme separated therefrom was decompressed and concentrated in a vacuum concentrator, dried by spraying and drying with a hot air dryer, and then pulverized with a particle size of 60 mesh to obtain a dry powder.
  • the grains, seeds and the culture solution of Aranicola proteoriticus prepared above or the dry powder of arazyme separated therefrom were combined in the following ratio to prepare a conventional method.
  • Cereals (30 parts by weight brown rice, 15 parts by weight brittle, 20 parts by weight of barley),
  • Seeds (7 parts by weight perilla, 8 parts by weight black beans, 7 parts by weight black sesame seeds),
  • a beverage comprising a culture solution of Aranicola proteoricicus or arazyme isolated therefrom was prepared as follows.
  • Substances such as liquid fructose (0.5 parts by weight), oligosaccharides (2 parts by weight), sugar (2 parts by weight), salt (0.5 parts by weight), water (75 parts by weight), and a culture medium of Aranicola proteoricicus or After the separate arazyme (0.5 parts by weight) homogeneously blended for instant sterilization, it was packaged in small packaging containers such as glass bottles and plastic bottles to prepare a healthy beverage.
  • aranicoim culture or arazyme isolated therefrom was added to 1,000 ml of a juice of a vegetable such as tomato or carrot to prepare a vegetable juice for health promotion in a conventional manner.
  • aranico coli or arazyme isolated therefrom was added to 1,000 ml of fruit juice such as apples or grapes to prepare fruit juice for health promotion in a conventional manner.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Epidemiology (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Mycology (AREA)
  • Nutrition Science (AREA)
  • Food Science & Technology (AREA)
  • Polymers & Plastics (AREA)
  • Coloring Foods And Improving Nutritive Qualities (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention se rapporte à une composition pharmaceutique comprenant de l'arazyme en tant que principe actif pour la prévention ou le traitement de maladies métaboliques. En détail, on a trouvé que l'arazyme produite par Aranicola protéolyticus réduit les taux d'hémoglobine glyquée (HbA1c), le cholestérol total, le triglycéride et l'acide gras non estérifié (NEFA) dans le plasma de souris, améliore la tolérance au glucose dans un modèle animal alimenté par un régime riche en graisse sous une charge de glucose, stimule la libération et la synthèse d'insuline dans les cellules bêta pancréatiques, atténue la résistance à l'insuline dans les cellules hépatiques et supprime l'accumulation de lipides et la fibrose hépatique. Par conséquent, l'arazyme de la présente invention peut être utilisée de manière efficace dans une composition pour la prévention ou le traitement de maladies métaboliques.
PCT/KR2017/011095 2016-10-06 2017-10-02 Composition pharmaceutique comprenant de l'arazyme en tant que principe actif pour la prévention ou le traitement d'une maladie métabolique WO2018066969A1 (fr)

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KR100909998B1 (ko) * 2007-03-29 2009-07-29 주식회사 인섹트 바이오텍 아라자임을 유효성분으로 하는 유방암 예방 및 치료용약학적 조성물
KR100945960B1 (ko) * 2008-01-11 2010-03-05 주식회사 인섹트 바이오텍 아라자임을 유효성분으로 하는 관절염 예방 및 치료용조성물
JP2010514752A (ja) * 2006-12-28 2010-05-06 インセクト バイオテック カンパニー， リミティッド アラザイムを有効成分とする肝機能保護用薬学的組成物
KR101476399B1 (ko) * 2012-07-31 2014-12-24 주식회사 인섹트 바이오텍 아라자임을 유효성분으로 하는 아토피 피부염 예방 및 치료용 조성물

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KR100836711B1 (ko) * 2006-12-28 2008-06-10 주식회사 인섹트 바이오텍 아라자임을 유효성분으로 하는 간기능 보호용 약학적조성물
JP2010514752A (ja) * 2006-12-28 2010-05-06 インセクト バイオテック カンパニー， リミティッド アラザイムを有効成分とする肝機能保護用薬学的組成物
KR100909998B1 (ko) * 2007-03-29 2009-07-29 주식회사 인섹트 바이오텍 아라자임을 유효성분으로 하는 유방암 예방 및 치료용약학적 조성물
KR100945960B1 (ko) * 2008-01-11 2010-03-05 주식회사 인섹트 바이오텍 아라자임을 유효성분으로 하는 관절염 예방 및 치료용조성물
KR101476399B1 (ko) * 2012-07-31 2014-12-24 주식회사 인섹트 바이오텍 아라자임을 유효성분으로 하는 아토피 피부염 예방 및 치료용 조성물

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