KR101176618B1 - Composition comprising ajoene for preventing or treating a disease caused by overexpression of LXR-alpha - Google Patents

Abstract

 The present invention is to prevent disease caused by the expression or excessive activity of LXRα (Liver X recptorα) or SREBP-1 (Sterol Response Element Binding Protein-1) comprising azoene or a garlic extract fraction containing a large amount thereof as an active ingredient, A composition that can be used for improvement and treatment. The composition of the present invention can be effectively used for the prevention, improvement and treatment of fatty liver, hypertriglyceridemia, hyperreninemia, hypertension due to renin, aldosteroneism, adrenal protein dystrophy, renal glomerulosclerosis, proteinuria, nephropathy. In addition, the composition of the present invention can be used for the prevention and treatment of diabetes.

Description

Composition comprising ajoene for preventing or treating a disease caused by overexpression of LXR-alpha}

The present invention relates to the pharmaceutical or food use of ajoene or garlic extract fractions containing a large amount of azoene. More specifically, prevention of disease or diabetes due to excessive activity of LXRα (Liver X receptor-alpha) or SREBP-1 (Sterol Response Element Binding Protein-1) using azoene or garlic extract fractions containing a large amount of azoene , Improvement and treatment.

 The research for the present invention was supported in part by the Center for Excellence (ERC) [Task Unique Number: 2009-0063233 Task Name: Discovery of Metabolic Disease Control Drug Targets and Candidate Drug Activity Studies].

Liver X receptor (LXR) is a type of nuclear hormone receptor, a gene involved in cholesterol metabolism and homeostasis, such as apolipoprotein E (apoE), ABCA1, ABCG1, ABCG5, ABCG8, cholesterol 7α-hydrate Plays an important role in the transcriptional regulation of cholesterol 7α-hydroxylase and scavenger receptor class B type I genes [Schwarz et al . , Biochem . Biophys. Res . Commun ., 2000, 274: 794-802. LXR also acts directly on the SREBP-1c gene to regulate lipid metabolism [Yoshikawa et al . , Mol . Cell . Biol ., 2001, 21: 2991-3000.

 LXR isomers include LXRα and LXRβ. LXRα is mainly present in the liver and LXRβ is expressed mostly in organs. LXRα is activated by natural ligands, oxysterols, high concentrations of sugars and artificial ligands, T0901017, GW3965 and the like, and regulates the expression of genes responsible for lipid formation and cholesterol homeostasis in the body. LXRα acts as a lipid sensor in the production of liver lipids, which strongly increases the expression and activity of SREBP-1c, a key transcription factor that regulates the expression of lipid-forming genes, thereby promoting fatty acid synthesis in liver tissues and increasing blood triglyceride levels. .

 There are largely SREBP-1c dependent pathways and SREBP-1c independent pathways in which LXRα activation causes nonalcoholic fatty liver disease. In SREBP-1c-dependent fatty liver, the expression of lipogenetic genes is upregulated through transcriptional activation of LXRα-mediated SREBP-1c, and in SREBP-1c-independent fatty liver, LXRα activation increases the expression of CD36 protein, a transporter of free fatty acids. By stimulating the transport of fatty acids to the liver.

It has also been reported that LXRα plays an important role in the secretion of renin in the kidney. LXRα and LXRβ are both expressed in abundance in juxtaglomerular cells that produce renin. According to Morello et al., L09Rα agonists T0901017 or GW3965 increase the expression of renin mRNA in the kidney and increase blood renin activity [Mello et al . , J. Clin . Invest . , 2005, 115: 1913-1922. Excessive increase in blood renin leads to hyperreninemia, which leads to hypertension and aldosteroneism.

LXR also regulates the expression of the ABCD2 gene associated with adrenoleukodystrophy (ALD), a rare disease in which the body's `` very long chain fatty acid '' (VLCFA) does not break down and enters the brain and destroys nerve cells. It has been reported to be useful for the treatment of ALD [Winehopper et al . , J. Biol . Chem . , 2005, 280: 41243-41251.

 SREBP (Sterol Response Element Binding Protein) is a protein that binds to sterol response element (SRE), a transcriptional regulatory region of sterol-regulated genes, and exists in three isoforms. SREBP-1a and SREBP-1c are transcribed from the same gene and SREBP-2 is expressed from other genes. SREBP-1c is a gene mainly involved in fatty acid synthesis, and SREBP-2 is a transcription factor that regulates transcription of genes involved in cholesterol synthesis.

SREBP-1 and SREBP-2 increase in the expression of the kidneys with age, thereby increasing the lipid synthesis and accumulation of triglycerides and cholesterol in the kidneys, such as glomerulosclerosis, proteinuria, kidney It has been reported to play an important role in the nephropathy (Jiang et al ., Kidney Int ., 2005, 68 (6): 2608-2620).

SREBP is usually present as a endoplasmic reticulum membrane protein and is 125 kilodaltons (kDa) in size. Before being activated by stimulation such as sterol depletion, it is bound to the membrane in an inactivated form, and upon activation, it is transferred to the Golgi apparatus and cleaved into an active protein of 65 kilodalton size. When activated, SREBPs move into the nucleus and bind to the SRE of the target gene, thereby increasing the expression of liposynthetic genes. Target genes of SREBP-1c include fatty acid synthase (FAS), acetyl CoA carboxylase (ACC), and stearoyl CoA desaturase (SCD). If the amount of free fatty acids and fatty acids directly produced by the liver from the blood is greater than the amount of VLDL or beta-oxidized fatty acids, the lipid metabolism balance in the liver is broken down, leading to fatty liver disease. Therefore, SREBP-1c, which induces and regulates proteins such as FAS, ACC, and SCD, which are responsible for fat production in the liver, is an important factor of alcoholic or non-alcoholic steatosis [Kohjima et al . , Int . J. Mol . Med . , 2008, 21 (4): 507-511, Donohue, World J. Gastroenterol . 2007, 13 (37): 4974-4978.

 Fatty liver is a medical condition in which fat exceeds more than 5% of the total liver weight. Liver disease, including this, is known to be the second most serious disease after cancer in developed countries in the 40-50 adult population. About 30% of the population in major countries, including developed countries, already has fatty liver symptoms, and 20% of them progress to cirrhosis through liver fibrosis. Half of these cirrhosis patients die of liver disease within 10 years of diagnosis. Non-alcoholic fatty liver is currently increasing incidence due to increased dietary intake and Western insufficiency and lack of exercise, and it is recommended to improve lifestyles such as eating habits as a treatment.

 Currently, there are few drugs useful for treating fatty liver pharmacologically, and only exercise and diet are recommended. However, the treatment efficiency of fatty liver by these methods is very low, and there is an urgent need for the development of effective therapeutic agents. Betaine, glucuronate, methionine, choline, and lipotrophic agents are often used as adjuvant, but the medicinal evidence for them is fully demonstrated. It is not. Therefore, the development of a safe fatty liver treatment that does not cause side effects with excellent effects is urgently needed.

 Diabetes mellitus is a disease that can be caused by a lack of insulin action or due to a decrease in the body's responsiveness to insulin, resulting in decreased metabolic abnormalities, which leads to decreased glucose utilization in the cell and persistent hyperglycemia, and chronic vascular complications. Diabetes is divided into type 1 diabetes and type 2 diabetes, with more than 85% of diabetics belonging to type 2 diabetes and occurring mainly in adults. Type 2 diabetes can be accompanied by a decrease in the body's responsiveness to insulin and a disorder of insulin secretion from endocrine cells. Insulin resistance refers to a condition in which the reactivity of living cells or tissues to insulin at a given insulin concentration is less than normal. The inventors of the present invention have shown that oltipraz is effective in the treatment of diabetes due to obesity due to a high-fat diet and the resulting increase in insulin resistance (Registration No. 690818). Various diabetes treatments are currently being developed, but there is a need for the development of new drugs that are more effective.

 Ajoene, on the other hand, is a compound that can be separated from crushed or crushed garlic. The production of azoene from garlic involves the release of allinase, stored in garlic vacuoles, when the tissues are destroyed, converting allin to allylsulfenic acid and producing allicin from both molecules. Allicin is converted to azoene, a stable compound, by sulfoxide rearrangement.

 Pure separation of azoene from garlic is complicated and differs according to garlic materials, and the yield is low through several stages of separation. Therefore, organic synthesis can be used to secure large quantities. Azoene is a structurally stable compound which can be easily prepared by total synthesis using a low molecular organic compound as a starting material.

 Azoene has an allyl vinyl disulfide group, which is known to exert various physiological activities by sulfenylating sulfhydryl groups of various proteins. The physiological activities of azoene include anti-tumor, antibacterial, and platelet aggregation inhibitors (Prostaglandins Leukot Essent Fatty Acids, vol. 49, No. 2, p587-595, 1993), strengthening immunity, enhancing brain function, and removing hangover (public patent) Publication 2007-0017452).

However, it has not been reported that azoene can be used as a composition for the prevention and treatment of diseases, diabetes due to overexpression or overactivity of LXRα or SREBP-1.

In the present invention, it is an object of the present invention to find and provide a novel inhibitor of LXRα or SREBP-1. Diseases caused by overexpression or overactivity of LXRα or SREBP-1, such as fatty liver, hypertriglyceridemia, hyperreninemia, hypertension caused by renin, aldosteroneism, adrenal protein dystrophy, renal glomerulosclerosis, proteinuria, kidney damage, etc. It is also an object of the present invention to provide a pharmaceutical or food composition for the prevention, amelioration and treatment. In addition, it is also a problem to be solved by the present invention to provide a composition for preventing or treating diabetes.

The present invention provides a pharmaceutical composition or food composition comprising azoene or a garlic extract fraction containing a large amount as an active ingredient to solve the above problems.

 The composition comprising the azoene of the present invention or a garlic extract fraction containing a large amount thereof as an active ingredient can be effectively used for the prevention, improvement and treatment of diseases due to overexpression or overactivity of LXRα or SREBP-1. The composition includes fatty liver, hypertriglyceridemia, hyperreninemia, high blood pressure due to renin, aldosteroneism, adrenoleukodystrophy, glomerulosclerosis, proteinuria, nephropathy, and the like. It can be effectively used for the prevention, improvement and treatment of the. In addition, the composition of the present invention can be used to prevent and treat diabetes. The composition of the present invention can be used to lower the level of blood sugar that is increased with a high lipid diet.

The composition of the present invention can be used for the manufacture of medicines or nutraceuticals that can be used for the prevention, improvement or treatment of the disease.

FIG. 1 is a result of treating T0901317, an LXRα activator, with HepG2, a hepatocyte cell line, and observing the effect of Ajoen on the expression level of LXRα. (Con: no treatment group, **: p <0.01 compared with Con group, #: p <0.05 compared with group treated with T0901317 only)
FIG. 2 shows the results of treatment of hepatic cell line HepG2 with LXRα activator T0901317 and the effect of Ajo treatment on LXRE. (Con: untreated group, **: p <0.01, compared with Con group) #: P <0.05 compared with T0901317 only, ##: p <0.01 compared with T0901317 only)
FIG. 3 is a result of treating T0901317, an LXRα activator, with HepG2, a hepatocyte cell line, and observing the effect of Ajoen on the expression level of SREBP-1 protein. (Con: no treatment group, **: p <0.01 compared with Con group, #: compared with group treated with only T0901317 p <0.05, ##: p <0.01 compared with group treated with only T0901317)
Figure 4 shows the results of observing the effect of ajoen treatment (Ajo) on the hepatic expression level of the lipid-generating gene LXR induced by a high-fat diet. RXRα or SREBP-1 mRNA levels in the normal diet were expressed as relative values. (ND: normal diet HFD: high-fat diet Ajo: azoene **: p <0.01 compared to ND group, #: p <0.05 compared to HFD alone group, ##: p <0.01 compared to HFD alone group )
Figure 5 is a result of observing the effect of the administration of azoene (Ajo) on the content of triglycerides in liver tissue in a lipid diet-induced fatty liver animal model. (ND: Dietary HFD: High-fat diet Ajo: N **: p <0.01 compared to ND group, #: p <0.05 compared to HFD alone group, ##: p <0.01 compared to HFD alone group)
Figure 6 is a comparison of the fat liver animal model induced by a high-fat diet and liver tissue when azoen (Ajo) administered to the animal model stained by fat staining method (Oil Red O).
7 is a result confirming the effect of azoene treatment on body weight change in azoen (Ajo) administration schedule and a high-fat diet-induced fatty liver animal model. (ND: Dietary HFD: Dietary Ajo: Azoen, **: p <0.01 compared to ND group: p <0.05 compared to HFD alone group)
Figure 8 is a result of observing the effect of the administration of azoene on fasting blood glucose concentration in a high fat diet-induced fatty liver animal model. (ND: normal diet HFD: high-fat diet Ajo: azoene **: p <0.01 compared to ND group: p <0.05 compared to HFD alone group)

 The present invention relates to the pharmaceutical or food use of ajoene or garlic extract fractions containing a large amount thereof.

 The inventors of the present invention have repeatedly studied the substances affecting the expression and activity of LXRα and SREBP-1, surprisingly increased LXRα and SREBP-1 when treated with hepatocytes T0901317, which is known to be an activator of LXRα. It was found to inhibit the expression and activity of (Fig. 1-3). In addition, the inventors of the present invention, the expression of LXRα and SREBP-1 in mice with increased expression in a high-lipid diet was inhibited by azoene (Fig. 4), administration of a high-lipid diet increased the content of triglycerides in liver tissue When the azoene was administered to the mice, the content of the triglycerides was significantly decreased (FIG. 5), and the liver tissue was observed by lipo staining to confirm that the amount of fat was significantly suppressed by the administration of azoene ( 6). In addition, administration of azoene significantly reduced weight gain due to a high lipid diet (FIG. 7), and significantly decreased fasting blood glucose concentrations in a high fat diet induced fatty liver animal model (FIG. 8).

 Based on the above experimental results, the present invention provides a pharmaceutical composition for the prevention, improvement and treatment of diseases caused by overexpression or activity of LXRα or SREBP-1 containing azoene or a garlic extract fraction containing a large amount thereof as an active ingredient or Provide food compositions.

Compositions of the present invention are hypertriglyceridemia, hyperreninemia, renin-induced hypertension, aldosteroneism, adrenal protein dystrophy, renal glomerulosclerosis, proteinuria, kidney damage It may be used for the prevention, improvement or treatment, but is not limited thereto.

 Administration of the composition of the present invention inhibits the expression and activity of SREBP-1, a key transcription factor that regulates the expression of lipid generating enzyme genes through the regulation of LXRα activation, and furthermore, fatty liver disease caused by metabolic disorders through inhibition of the expression of lipid producing genes. Since it inhibits the accumulation of triglycerides in liver tissue due to, the composition of the present invention containing it as an active ingredient can be usefully used to prevent and treat fatty liver (liver steatosis).

 In addition, the present invention provides a composition for lowering the level of blood glucose increased by a high-fat diet containing azoene or a garlic extract fraction containing a large amount thereof as an active ingredient.

 The present invention provides a composition for preventing, improving and treating diabetes mellitus containing azoene or a garlic extract fraction containing a large amount thereof as an active ingredient.

 Furthermore, the present invention provides a method for preventing, ameliorating, and treating diabetes mellitus due to the overexpression or excessive activity of LXRα or SREBP-1 or blood glucose increase due to a high-lipid diet by administering azoene or a garlic extract fraction containing a large amount thereof. to provide.

 Hereinafter, the present invention will be described in detail.

The azoene used in the present invention can be produced or separated from garlic by a known method or obtained by synthesis. For example Hunter et al. [ Bioorg Med Chem Lett ., 2008, 18: 5277-5279], and can be synthesized and separated from garlic by the following method.

Specifically, for example, it is possible to manufacture through the following process. Crushed or ground garlic or these filtered filtrates are left at room temperature or at ~ 37 ^o C for at least 12 hours to allow the garlic alliane to pass through allicin to produce azoene, and then add polar solvents such as methanol and ethanol for 1 hour. To 7 days, preferably 24 hours to 72 hours, and extracted under 10 to 100 ℃ concentrated under reduced pressure. The concentrate is dispersed in water and fractionated with a solvent such as diethyl ether. From the obtained fraction, an azoene-rich fraction can be obtained through silica gel chromatography, and further chromatography can be performed to separate pure azoene, but not limited thereto.

 Azoene, ie, (E. Z) -4,5,9-trithiadodeca-1,6,11-triene-9-oxide ((E, Z) -4,5,9-trithiadodeca-1, 6,11-triene-9-oxide) has the geometric isomers of the trans form (E- azoene) and cis type (Z-azoene). The azoene contained in the composition of the present invention corresponds to azoene which is a cis type, a trans type, or a mixed form thereof.

The azoene or garlic extract fraction containing a large amount of the present invention may be prepared by pharmaceutically acceptable salts and solvates according to conventional methods in the art. As the pharmaceutically acceptable salt of the present invention, acid addition salts formed by free acid are useful. Acid addition salts are prepared by conventional methods, for example by dissolving a compound in an excess of aqueous acid solution and precipitating the salt using a water miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. Equivalent molar amounts of the compound and acid or alcohol (eg, glycol monomethyl ether) in water can be heated and the mixture can then be evaporated to dryness or the precipitated salts can be suction filtered. In this case, organic acids and inorganic acids may be used as the free acid, hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid, etc. may be used as the inorganic acid, and methanesulfonic acid, p -toluenesulfonic acid, acetic acid, trifluoroacetic acid, Citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid ( gluconic acid), galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanic acid, hydroiodic acid, and the like.

 In addition, bases can be used to make pharmaceutically acceptable metal salts. An alkali metal or alkaline earth metal salt is obtained by, for example, dissolving a compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and then evaporating and drying the filtrate. At this time, as the metal salt, it is particularly suitable to prepare sodium, potassium or calcium salt, and the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (for example, silver nitrate).

Pharmaceutically acceptable salts of the azoene or garlic extracts containing the same include salts of acidic or basic groups which may be present in the compound, unless otherwise indicated. For example, pharmaceutically acceptable salts include sodium, calcium and potassium salts of the hydroxy group, and other pharmaceutically acceptable salts of the amino group include hydrobromide, sulfate, hydrogen sulphate, phosphate, hydrogen phosphate, dihydrogen Phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate) and p -toluenesulfonate (tosylate) salts, and methods or processes for preparing salts known in the art It can be prepared through.

 The composition comprising the azoene of the present invention or a garlic extract fraction containing a large amount thereof may further include appropriate carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

 The composition of the present invention comprises 0.01 to 99.9% by weight, preferably 0.1 to 90% by weight, more preferably 0.1 to 50% by weight of azoene or a garlic extract fraction containing a large amount thereof. However, the composition is not limited thereto, and may vary depending on the condition of the patient, the type of disease, and the progress of the disease.

 Compositions comprising azoene or garlic extract fractions containing a large amount thereof according to the present invention, powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc. Carriers, excipients and diluents which may be formulated in the form of suppositories and sterile injectable solutions, and the carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia Rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil Can be. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, sucrose, or the like in the extract or compound. (sucrose), lactose (lactose), gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As a base material of suppositories, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

 The preferred dosage of the composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the composition comprising the azoene or garlic extract containing the same as an active ingredient of the present invention is 0.01 mg / kg to 10 g / kg per day, preferably 1 mg / kg to 1 g / kg It is recommended to administer. The administration may be carried out once a day or divided into several doses. Accordingly, the dosage is not limited in any way to the scope of the present invention.

 The composition of the present invention may be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

 The composition of the present invention can be used as a pharmaceutical composition or a composition for nutraceuticals. Therefore, the present invention is a health functional food comprising at least one selected from the group consisting of azoene, garlic extract fractions containing a large amount of azoene, their food acceptable salts, solvates and hydrates thereof. And drinks. Foods to which the azoene or garlic extract fraction containing a large amount of the present invention can be added include, for example, various foods, beverages, gums, teas, vitamin complexes, health supplements, and the like, powders, granules, and tablets. , Capsules or beverages.

 At this time, the amount of the compound in the food or beverage is generally 0.01 to 15% by weight of the total food weight of the health food composition of the present invention, and 0.02 to 10 g, Can be added in a proportion of 0.3 to 1 g. The health beverage composition of the present invention, in addition to containing the compound as an essential ingredient in the indicated proportions, has no particular limitation on the liquid component and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. Examples of the above-mentioned natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin and the like Sugar, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

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

 Azoene or garlic extract fractions containing a large amount of the present invention is a drug that can be used with confidence even for long-term use for the purpose of prevention and improvement because there is little toxicity and side effects.

Hereinafter, the present invention will be described in detail with reference to the following examples and experimental examples. However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the contents of the present invention are not limited by the following Experimental Examples.

Example  1. from garlic Azoen  Produce

 2 kg of fresh crushed garlic was stored at room temperature for 12 hours and at 37 ° C. for 5 hours, and 80% ethanol was added for 24 hours at room temperature. The extract was concentrated under reduced pressure, dispersed in water and fractionated with diethyl ether. 5.2 g of the ether solution fraction was subjected to silica gel column chromatography using a gradient solvent mixture with n-hexane / ethyl acetate and n-hexane / acetone to obtain 120 mg of a small fraction rich in azoene (ARF). 53 mg of pure azoene was obtained from reverse fractions by reversed phase high performance liquid chromatography (μ-Bondapak C-18, 10 × 300 mm; 70% methanol; 2.0 ml / min; ultraviolet 254 nm). The chemical structure of azoene was confirmed by mass spectrometry and nuclear magnetic resonance spectroscopy as a (Z) -ajoene isomer.

Example  2. By organic synthesis Azoen   Produce

The synthesis of azoene was prepared by a method slightly converted from an organic synthesis method recently reported by Hunter et al. (Bioorg Med Chem Lett., 2008, 18: 5277-5279). In other words, allyl thiol is used as a starting material, and allylpropargyl sulfide is obtained through propargylation, and thiol acetic acid is substituted with azobisisobutyronitrile. Regioselective radical addition was carried out in the presence of (azobisisobutyronitrile, AIBN) to synthesize a vinyl thiol acetate (vinylthioacetate). The thiol acetate obtained was subjected to vinyl disulfide through deprotection and sulfenylation using S-allyl p-toluenesulfonylthioate. Azoene was synthesized by oxidizing it with meta-chloroperoxybenzoic acid (m-CPBA). The chemical structure of the synthesized azoene was confirmed by mass spectrometry and nuclear magnetic resonance spectroscopy as (Z) -ajoene isomer.

Reference Example  1: Experimental Animals and Diet

Male C57BL / 6 mice (average weight 25-30 g) used as experimental animals were purchased from Charles River Orient (Seoul. Korea). Before the experiment, animals were adapted from the Seoul National University College of Pharmacy Animal Experimental Research Building with controlled humidity of 55 ± 5%, temperature of 22 ± 2 ℃ and ventilation for more than 1 week. Changed the contrast in time periods. No significant changes were observed in the amount of food and water for the duration of the experiment. The weight and condition of the animals were checked once weekly and raised for 8 weeks on a high-fat diet (Dyets Inc., Bethlehem) or normal diet and administered azoene (10 or 30 mg / kg, 5 times / week) for the last 4 weeks. It was. Each group consisted of a total of eight mice.

Reference Example  2: Sample Preparation

A high-fat diet for fatty liver induction was purchased from Diet Co., USA. Desired concentrations of azoene were prepared by dilution with 40% PEG # 400 [PEG # 400: water = 4: 6].

Reference Example  3: Real time - RT PCR

CDNA was obtained using total RNA (2 μg), d (T) ₁₆ primer and AMV reverse transcriptase extracted from mouse liver. Relative amounts of genes were quantified by Realtime RT-PCR method using CyBr green dye. Realtime RT-PCR used Light-cycler ^2.0 from Roche (Mannheim, Germany). PCR was performed according to the manufacturer's method and the relative amount of each gene was analyzed using the Light-cycler software 4.0 program.

Reference Example  4: Western Blot

Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using a Mighty Small II SE 250 apparatus according to the Laemmli UK method (1970). Dissolve the fraction of liver sample in sample dilution buffer [63mM Tris (pH.6.8), 10% glycerol, 2% SDS, 0.0013% bromophenol blue, 5% β-mercaptoethanol] and use 7.5%, 9% gel Electrophoresis was performed in buffer (including Tris 15g, glycine 72g, SDS 5g in 1L solution). The electrophoresis gel was transferred to nitrocellulose paper for 1 hour at 190 mAmps in transition buffer [25 mM Tris, 192 mM glycine, 20% v / v methanol (pH.8.3)] using a transfer electrophoresis device. After reacting Anti-SREBP-1 as a primary antibody, Horse radish peroxidase-conjugated goat anti-rabbit IgG was reacted for 1 hour with a secondary antibody and developed using an ECL chemiluminecence system (Amersham, Gaithesberg, MA). The homogeneity of the protein content in the samples was confirmed using anti-β-actin antibodies (Sigma, St. Louis, MO).

Reference Example  5: analysis method

The data presented in the experiments were analyzed using a pharmacological calculation program. The significance of the various experimental groups was analyzed by one-way square deviation analysis (Fisher, RA, Statistical). Methods for Research Workers , Edinburgh : Oliver & Boyd , 1925) The test was followed by the Newman-Kels test (Norman GR et al., Biostatistics: The Bare Essentials, 2000) ( ^{*, #} p <0.05, ^{**, ##} p <0.01).

Experimental Example  One: Azoen  By treatment LXR Expression inhibitory effect of α

 As a cell line, HepG2, a representative human liver cell line, was used. HepG2 was pretreated with azoene (Ajo, 10 or 30 μM) for 1 hour, treated with LXRα activator T0901317 (10 μM) for 12 hours, mRNA was isolated from hepatocytes, and cDNA was synthesized via RT-PCR. The primers (Human LXR, 5'-GATCGAGGTGATGCTTCTGGAG-3 '(sense) and 5'-CCCTGCTTTGGCAAAGTCTTC-3' (antisense)) were analyzed by real-time PCR.

When the LXRα activator T0901317 was treated with hepG2, a hepatocyte cell line, the mRNA expression of LXRα was markedly increased, and the relative mRNA level was 3 times, and the azoene treatment resulted in 1.9 times in 10uM and 1.8 times in 30uM. Suppressed (FIG. 1).

Experimental Example  2: Azoen  By treatment LXR α activity inhibition ( LXRE Binding capacity  Suppression effect

LXRα regulates gene expression by binding to a specific region (LXRE) present in the target gene promoter. A reporter gene assay (Luciferase reporter gene assay) was carried out to observe whether the change in LXRE binding capacity by azoene treatment.

 First, a vector containing LXRE was transfected to a HepG2 cell line using lipofectamine (Invitrogen, USA). After 12 hours, HepG2 was pretreated with azoene (Ajo, 10 or 30 μM) for 1 hour and treated with LXRα activator T0901317 (10 μM) for 12 hours. Then, using a passive lysis buffer (Promega, USA) to obtain a fraction of the cells, Luciferase activity was measured using a Luminometer.

The luciferase activity, which was markedly increased by 2.3 times relative luciferase activity by T0901317 treatment, was 1.8 times in azoene 10uM and 1.3 times in 30uM by azoene pretreatment, which was statistically significantly inhibited (FIG. 2). ). This suggests that azoene has an effect of inhibiting the activity of LXRα.

Experimental Example  3: Azoen  By treatment SREBP -1 expression and activity inhibitory effect

 T0901317, an LXRα activator, was treated with HepG2, a hepatocyte cell line, and T091317 and azoene, and the mRNA and protein expression levels of SREBP-1 were compared (Fig. 3). HepG2 was pretreated with azoene (Ajo, 10 or 30 μM) for 1 hour and treated with LXRα activator T0901317 (10 μM) for 12 hours. Then, the protein was separated and Western blot was performed to observe the protein change of SREBP-1 (Fig. 3, bottom), mRNA was isolated to synthesize cDNA via RT-PCR, and then a specific primer (human SREBP-1) , 5'-CGACATCGAAGACATGCTTCAG-3 '(sense) and 5'-GGAAGGCTTCAAGAGAGGAGC-3' (antisense)) were analyzed by real-time PCR (Figure 3, top).

When the LXRα activator (T0901317) was treated in the hepatocyte cell line HepG2, mRNA (S3, top) and protein expression (FIG. 3, bottom) of SREBP-1, a cell protein that regulates lipid production gene expression, were compared with the control group in 12 hours. In comparison, the relative mRNA level was significantly increased by 4.8-fold (p <0.01), and the concentration-dependent increase was suppressed by azoene treatment at 2.8-fold at 10uM and 2.4-fold at 30uM.

Experimental Example  4: On a high-fat diet  Manifestation Increased LXR for α Azoen  Effect of kill

Effects of Azoene on Increased Expression of LXRα Induced by High Fat Diet Observed. The expression level of the intracellular lipid sensor LXRα was analyzed by real-time PCR using a specific primer after cDNA synthesis by mRNA isolation and RT-PCR in liver tissue. Expression of LXRα in liver tissues of mice fed with high-fat diet or normal diet for 8 weeks and fed azoene (10 or 30 mg / kg, 5 times / week) for the last 4 weeks in group fed with high-lipid diet The degree was measured. After separating mRNA from liver tissue and synthesizing cDNA through RT-PCR, real using specific primers (mouse LXR, 5'-TGCCATCAGCATCTTCTCTG-3 '(sense) and 5'-GGCTCACCAGCTTCATTAGC-3' (antisense)) Analyzed by -time PCR. When the level of LXRα mRNA of the normal diet group (ND) was set to 1, the relative LXRα mRNA level of the high lipid diet group or the high lipid diet + azoene administration group was shown (left of FIG. 4). The expression of LXRα, an intracellular lipid sensor, was significantly increased by 2.1 times relative mRNA level (p <0.01), and the increase in LXRα expression was 1.3-fold at 30 mg / kg of azoene with azoene. It was found to be suppressed.

Experimental Example  5: On a high-fat diet  Manifestation Increased SREBP For -1 Azoen  Effect of kill

 The expression of SREBP-1, a transcription factor that regulates the expression of lipogenesis enzyme genes, was also increased by a high-lipid diet and the effects of azoene administration were observed.

Expression level of SREBP-1 was measured in liver tissue of mice bred in the same manner as in Experimental Example 4. After separating mRNA from liver tissue and synthesizing cDNA through RT-PCR, using specific primers (mouse SREBP-1, 5'-AACGTCACTTCCAGCTAGAC-3 '(sense) and 5'-CCACTAAGGTGCCTACAGAGC-3' (antisense)) Was analyzed by real-time PCR. When the level of SREBP-1 mRNA of the normal diet group (ND) is 1, the relative SREBP-1 mRNA levels of the high lipid diet group and the high lipid diet + azoene administration group are shown on the right side of FIG. The expression of SREBP-1 mRNA was significantly increased (p <0.01) by the high-lipid diet (p <0.01), and the increase of this expression was doubled at 10 mg / kg azoene and 30 mg / kg by azoene administration. 1.6-fold significantly inhibited.

Experimental Example  6: On a high-fat diet  Accumulated Liver tissue  For the content of triglycerides in Azoen  Effect of kill

The content of triglycerides in liver tissue is an indicator of fatty liver. Normal and high lipid diets were administered to mice for 8 weeks and azoene was administered 5 times per week, 10 or 30 mg / kg, respectively, for the last 4 weeks. Observed. In the 8-week high-fat diet mice, triglyceride content in the liver tissue was 61 mg / g liver, which was significantly increased compared to 27 mg / g liver in the normal diet group, whereas 10 mg / kg was administered when azoene was administered. When 36 mg / g liver and 30 mg / kg were administered, the content of triglyceride in the tissue was significantly decreased to 31 mg / g liver (FIG. 5).

Experimental Example  7: On a high-fat diet  Induced Fatty Liver Animal Model Liver tissue  About Azoen  Effect of dosing

The treatment effect of fatty liver induced by high-lipid diet by the administration of azoene was analyzed using Oil Red O staining, a fat-specific dye. Liver tissue obtained in Experiment 6 was fixed with 10% neutral formalin solution, and the tissue was embedded with paraffin after the usual fixation and dehydration procedures. The embedded tissues were tissue sections with a thickness of 4 μm, stained with oil red o, and observed with an optical microscope. As a result, in the high lipid diet group, the red stained area was markedly marked with oil red o (HFD + vehicle), and the red stained area was significantly decreased in the azoene-administered group (HFD + Ajo). The effect is the same as for (FIG. 6).

Experimental Example  8: On a high-fat diet  For weight gain caused Azoen  Effect of kill

Body weights of mice fed with high-fat diet (Dyets Inc., Bethlehem) or normal diet for 8 weeks and administered azoene (10 or 30 mg / kg, 5 times / week) for the last 4 weeks were measured during the breeding period. . Each group consisted of a total of eight mice. Administration of azoene 30mg / kg significantly reduced the weight gain due to a high-fat diet (Fig. 7) it can be seen that there is a prevention and treatment effect of obesity.

Experimental Example  9: On a high-fat diet Increased  For blood glucose levels Azoen  Effect of kill

Fasting 0.01 ml of fasting blood from the tail vein of mice fed either high fat diet (Dyets Inc., Bethlehem) or normal diet and given azoene (10 or 30 mg / kg, 5 times / week) for the last 4 weeks. The glucose concentration in plasma was measured by Accucheck blood glucose meter (Roche). The blood sugar level was significantly increased due to the high quality diet (162 mg / dl), compared with the normal dietary blood concentration of 68 mg / dl (102 mg / dl, 10 mg / kg and 30 mg / kg). Fasting blood glucose concentrations increased with a high lipid diet at 92 mg / dl (FIG. 8).

The following describes an example of the formulation of the composition containing the azoene or garlic extract fraction containing a large amount of azoene of the present invention, the present invention is not intended to limit it, but is intended to explain in detail only.

Formulation example  One. Sanje  Produce

Azoene 20 mg

Lactose 100 mg

Talc 10 mg

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

Formulation example  2. Preparation of tablets

10 mg of azoene

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

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

Formulation example  3. Preparation of capsules

Azoen 10 mg

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2 mg

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

Formulation example  4. Preparation of injections

Azoen 5 mg

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

Na ₂ HPO ₄ -12H ₂ O 26 mg

(2 ml) per 1 ampoule according to the usual injection preparation method.

Formulation example  5. Liquid  Produce

Azoene-rich garlic extract (ARF) 20 mg

10 g of isomerized sugar

5 g of mannitol

Purified water quantity

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

Formulation example  6. Manufacture of dietary supplements

Garlic extract (ARF) containing a large amount of azoene 1000 mg

Vitamin mixture quantity

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

0.13 mg of vitamin B ₁

0.15 mg of vitamin B ₂

0.5 mg of vitamin B ₆

Vitamin B ₁₂ 0.2 g

Vitamin C 10 mg

10 μg biotin

Nicotinic Acid 1.7 mg

50 μg folic acid

Calcium Pantothenate 0.5mg

Mineral mixture

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dibasic calcium phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride 24.8 mg

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

Formulation example  7. Manufacture of health drinks

Azoene 100 mg

15 g of vitamin C

100 g of vitamin E (powder)

Iron lactate 19.75 g

3.5 g of zinc oxide

Nicotinamide 3.5 g

0.2 g of vitamin A

Vitamin B ₁ 0.25 g

Vitamin B ₂ 0.3 g

Water quantity

After mixing the above components in accordance with a conventional healthy beverage production method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and then refrigerated and stored in the present invention For the preparation of healthy beverage compositions.

 Although the compositional ratio is relatively mixed with a component suitable for a favorite drink, it is also possible to arbitrarily modify the compounding ratio according to the regional or national preference such as the demand class, the demanding country, and the use purpose.

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Claims (11)

delete Fatty liver, hypertriglyceridemia, comprising as an active ingredient at least one selected from the group consisting of azoene, garlic extract fractions containing azoene, pharmaceutically acceptable salts thereof, solvates thereof and hydrates thereof, A pharmaceutical composition for the prophylaxis or treatment of a disease selected from the group consisting of hyperreninemia, hypertension due to renin, aldosteroneism, adrenal protein dystrophy, renal glomerulosclerosis, proteinuria and nephropathy. Pharmaceutical composition for the prevention or treatment of fatty liver comprising at least one selected from the group consisting of azoene, garlic extract fraction containing azoene, pharmaceutically acceptable salts thereof, solvates thereof and hydrates thereof . The pharmaceutical composition according to claim 3, wherein the fatty liver is metabolic fatty liver. delete Lowering blood sugar levels increased by a high-fat diet containing at least one selected from the group consisting of azoene, garlic extract containing azoene, pharmaceutically acceptable salts thereof, solvates thereof and hydrates thereof Pharmaceutical composition for. The pharmaceutical composition according to any one of claims 2 to 4 and 6, wherein the azoene is separated from garlic or prepared by organic synthesis. The garlic extract fraction containing azoene is extracted with pulverized garlic with a polar solvent, concentrated under reduced pressure and dispersed in water to obtain a diethyl ether solvent. A pharmaceutical composition, characterized in that it is fractionated and separated by silica gel chromatography. delete Fatty liver, hypertriglyceridemia, comprising as an active ingredient at least one selected from the group consisting of azoene, garlic extract fractions containing azoene, their food acceptable salts, solvates thereof and hydrates thereof, A health functional food for the prevention or amelioration of diseases selected from the group consisting of hyperreninemia, hypertension due to renin, aldosteroneism, adrenal protein dystrophy, renal glomerulosclerosis, proteinuria and kidney damage. The dietary supplement of claim 10 in the form of a powder, granule, tablet, capsule, syrup or beverage.

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