JP6153241B2 - Liver dysfunction prevention and improvement agent - Google Patents

Description

  The present invention relates to an agent for preventing and improving liver dysfunction containing ozonized fats and oils, a food and drink for preventing and improving liver dysfunction, and a feed for preventing and improving liver dysfunction.

  In recent years, metabolic syndrome (visceral fat syndrome) has attracted attention as a pathological condition that develops in relation to lifestyle habits. Metabolic syndrome refers to a condition with two or more risk factors of hyperglycemia, hypertension, and lipid abnormalities (high triglyceremia, low HDL cholesterolemia) in addition to visceral fat obesity, cerebral infarction, Since it increases the risk of the onset of arteriosclerotic diseases such as cerebral hemorrhage, ischemic heart disease, aortic aneurysm, prevention and improvement thereof is an important issue. Hyperglycemia, hypertension, and lipid abnormalities, which are risk factors for metabolic syndrome, are not independent of each other and are widely recognized as being related to each other. Obesity due to fat accumulation is a complication such as diabetes. It has become clear that this is one of the causes. According to recent research, stress caused by overeating, lack of exercise, and diversification of lifestyle habits can lead to modulation and failure of the body's defense response, resulting in chronic inflammation with prolonged low-level inflammatory conditions. It is also pointed out that chronic inflammation that causes irreversible organ dysfunction is the cause of the onset and progression of metabolic syndrome, arteriosclerotic diseases such as atherosclerosis, non-insulin dependent diabetes mellitus, lipid abnormalities, cancer, etc. It is attracting attention as being. In addition, inflammatory changes that occur in adipose tissue due to the accumulation of visceral fat due to obesity have been reported, and the influence on complications associated with obesity has been pointed out.

  It has been elucidated that adipocytes are involved in the regulation of biological functions such as homeostasis and energy metabolism by secreting various physiologically active substances called adipocytokines. Adiponectin and leptin to improve, TNFα to promote arteriosclerosis, plasminogen activator inhibitor-1 (PAI-1), IL-6, MCP-1, angiopoietin-like protein 2 (Angptl2), heparin A binding epidermal growth factor-like growth factor (HB-EGF) and the like are known. In obese adipose tissue, chronic inflammation accompanied by infiltration of inflammatory cells such as macrophages occurs, abnormal secretion of adipocytokines occurs, and adiponectin that works to improve insulin resistance is decreased. Such disruption of regulation by adipocytokines causes insulin resistance throughout the body and contributes to the development of complications such as diabetes. Adipocytokines play an important role in the pathogenesis of metabolic syndrome. It is believed that Such chronic inflammation is serious when accumulation of visceral fat, especially liver fat, occurs.

  As for the prevention and treatment of liver dysfunction or lifestyle-related diseases / obesity, conventionally, an agent for improving obesity / fatty liver prevention, visceral fat / liver lipid accumulation inhibitor (see Patent Document 1), potassium alginate as an active ingredient, cha Body fat reduction promoter, lipid metabolism promoter, preventive / therapeutic agent for liver disorders such as fatty liver, antifeedant (see Patent Document 2), soy sauce cake or its Numerous techniques such as PPAR activator, fatty acid metabolism activator, body fat burning promoter, obesity inhibitor, fatty liver inhibitor, anti-diabetic agent (see Patent Document 3), etc., containing an extract as an active ingredient are known. ing.

  Fatty acids are important substances as biological energy sources or biosynthetic biosynthetic materials, and are known to exhibit various physiological activities depending on the number of carbon atoms and the presence or absence of double bonds. As an active ingredient, a liver function improving agent (S2U type (S is a saturated fatty acid residue having 20 to 24 carbon atoms, U is an unsaturated fatty acid residue having 16 or more carbon atoms) triglyceride) Patent Document 4), a liver triglyceride concentration-reducing agent containing fish oil (see Patent Document 5), a therapeutic agent for diseases caused by accumulation of body fat containing punicic acid or its ester as an active ingredient (patent Reference 6), blood neutral fat lowering agent, liver neutral fat synthesis inhibitor (see Patent Reference 7), medium chain fatty acid and / or 5,11,14-eicosatrienoic acid as an active ingredient Body fat / subcutaneous fat reducing agent comprising a glycerin fatty acid ester containing a chain fatty acid as an active ingredient, an agent for preventing and / or improving obesity-derived diseases such as diabetes, hypertension, hyperlipidemia, fatty liver and gout (see Patent Document 8) ) Etc.

  Ozonated fats and oils obtained by converting oleic acid and other fats and oils that contain a large amount of oleic acid have been manufactured in Europe for a long time and used as therapeutic agents for trauma, etc., and many methods have been developed for efficient and stable production. (See Patent Documents 9 and 10). Ozonated oils and fats have been shown to exhibit various physiological activities such as antibacterial action, antioxidant action, antitumor action, and anti-inflammatory action by recent years, such as infections, ulcers, scabs, burns and others. Technology for use in medicine, beauty, feed or food additives by promoting action by combining ozonized oils and fats for topical treatment of tissue lesions of gastrointestinal tract, acne, skin care, etc. with thioctic acid (patent document) 11) and the like. However, it is not known that ozonized fats and oils are effective in the pathology of metabolic syndrome and effective in preventing and improving liver dysfunction.

JP 2011-021012 A JP 2009-185004 A JP 2009-242382 A JP 2008-247771 A JP 2008-184429 A JP 2008-150307 A JP 2006-232711 A International Publication No. 2004/022049 U.S. Pat. No. 2,857,410 US Pat. No. 5,183,911 US Patent Application Publication No. 2005/0113441

  An object of the present invention is to provide an agent for preventing and improving liver dysfunction, a food and drink for preventing and improving liver dysfunction, and a feed for preventing and improving liver dysfunction, which can be easily prepared and contains a commonly used substance as an active ingredient. .

  As a result of comparing the ozonized oil administration / non-administration groups using obese model rats, the present inventors found that (i) body weight and white adipose tissue amount were hardly different between the two groups. (Ii) Liver fat mass, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and serum plasminogen activity, which are factors deeply associated with liver dysfunction due to obesity As for the blood concentration of plasminogen activator inhibitor-1 (PAI-1), a new finding was found that a remarkable decrease was observed in the ozonized oil administration group, and the present invention was completed.

That is, the present invention
(1) a preventive / ameliorating agent for liver dysfunction caused by metabolic abnormalities characterized by containing ozonized olive oil as an active ingredient;
(2) and prevention improving agent according to (1) used in order to suppress the increase in the concentration of plasminogen activator inhibitor in the blood (PAI-1),
(3) The preventive / ameliorating agent according to (1) or (2) above, which is used as a health functional food ,
(4) The preventive / ameliorating agent according to (1) or (2) above, which is used as a feed additive .

  The agent for preventing or improving liver dysfunction according to the present invention suppresses liver fat accumulation, that is, suppresses accumulation of triglyceride in the liver, and can selectively release only triglyceride among lipids into the blood. Since it has the effect of reducing or increasing the concentration of PAI-1 in the liver, as another aspect of the present invention, as an aspect of the present invention, the liver fat accumulation-inhibiting or reducing agent containing ozonized oil and fat, the liver triglyceride accumulation containing ozonized oil and fat In addition to a suppressor or reducer, a decrease or increase inhibitor of blood PAI-1 concentration containing ozonated oils and fats, there can be mentioned food and drink or feed for liver fat accumulation inhibition or reduction containing ozonized oils and fats.

  According to the present invention, it is possible to prevent or improve symptoms of liver dysfunction caused by metabolic abnormalities. Moreover, the pathogenesis and progression of metabolic syndrome can be suppressed. Specifically, the accumulation of triglycerides in the liver can be suppressed, and only triglycerides among lipids can be selectively released into the blood, and the effect of reducing or increasing the concentration of PAI-1 in the blood is obtained. It is done. In addition, blood concentrations of AST and ALT, which are markers for liver dysfunction, can be reduced.

It is a figure which shows the result of having performed the single regression analysis on the relationship between the triglyceride density | concentration in a test rat, serum protein density | concentration, and a liver dysfunction marker. As triglycerides, liver triglyceride concentration (mg / g), serum triglyceride concentration (mg / dL). Serum proteins include serum PAI-1 concentration (ng / mL), serum C-reactive protein (CRP) concentration (pg / mL), serum adiponectin concentration (μg / mL), serum monocyte chemotactic factor 1 (MCP) -1) Concentration (pg / mL). Aspartic acid aminotransferase (AST) activity (IU / L) was used as a liver dysfunction marker.

  In the present invention, the ozonized oil / fat is a substance obtained by conducting ozone to an oil / fat containing an unsaturated fatty acid, and means an oil / fat containing trioxolane produced by the ozonization of a double bond of an unsaturated fatty acid. Ozonated fats and oils can be prepared by storing liquid fats and oils containing unsaturated fatty acids in a container and conducting an ozone / oxygen mixed gas at room temperature at a flow rate of about 1 to 5 L / min while stirring the fats and oils. it can. The oil or fat used as the raw material is preferably an oil or fat containing oleic acid, linoleic acid, or linolenic acid, which are unsaturated fatty acids, and oils and fats that are liquid at room temperature containing oleic acid in a high proportion are particularly suitable. Examples of such fats and oils include vegetable oils and edible oils such as olive oil, rapeseed oil, safflower oil, and sunflower oil. When oil and fat that are liquid at room temperature are used, ozonized oil is obtained, and when olive oil is used, ozonized olive oil is obtained. Moreover, it is preferable to use what distilled the impurity as fats and oils, and a solvent of fats and oils can use dichloromethane, chloroform, carbon tetrachloride, cyclohexane, methanol, ethyl acetate, etc. The ozone / oxygen mixed gas to be conducted can be prepared by a known production method such as a method using silent discharge of gaseous oxygen, a method using ultraviolet irradiation, or a method of electrolyzing dilute sulfuric acid.

  The agent for preventing and improving liver dysfunction of the present invention is used for preventing the onset of liver dysfunction, reducing symptoms of dysfunction, and improving symptoms such as healing. Causes of liver dysfunction include metabolic abnormalities, alcohol intake, viral infections, immune abnormalities, genetic factors, and the like. The present invention is particularly used for preventing and improving liver dysfunction caused by metabolic abnormalities. Liver dysfunction to prevent and improve can include liver failure with signs and symptoms such as liver failure, jaundice, hepatomegaly, ascites accumulation, portal hypertension, hepatic encephalopathy, cholestasis, Specifically, diseases such as fatty liver, cirrhosis, acute hepatitis and chronic hepatitis can be exemplified. The agent for improving and preventing liver dysfunction according to the present invention has an action of suppressing the accumulation of triglycerides in the liver, and is particularly effective in suppressing the accumulation of liver fat during obesity, and the onset of fatty liver and metabolic syndrome. Can be used to prevent or improve symptoms. The agent for preventing or improving liver dysfunction of the present invention can be used for reducing or suppressing the increase in blood PAI-1 concentration or for reducing blood AST or ALT concentration. PAI-1 is secreted from the liver, blood vessels and adipocytes due to inflammation, and the increase in blood PAI-1 concentration is not only increased in the metabolic syndrome, but also important as a thrombotic tendency and an aversion factor of vascular lesions, There are reports that it is related to the prevention and treatment of hepatitis or cancer. Therefore, ozonized oils and fats having an effect of improving and preventing liver dysfunction can also be used for the prevention and improvement of thrombosis and hepatitis, and the suppression of cancer growth and metastasis.

  The liver dysfunction preventive / ameliorating agent of the present invention can be formulated into a pharmacologically / pharmaceutically acceptable dosage form according to the administration route. The administration route may be either systemic administration or local route, but it is preferable to administer ozonized oils and fats via the intestinal tract by oral administration. The dosage form may be a tablet, capsule, granule, powder, liquid, syrup, or jelly preparation for oral administration, or an injection for direct administration to a body tissue / organ. Additives such as excipients, binders, disintegrants, dispersants, lubricants, coating agents, preservatives, colorants, flavoring agents, etc. may be used for the liver dysfunction prevention / amelioration agent. Alternatively, controlled release control may be performed.

  The administration method of the agent for preventing and improving liver dysfunction according to the present invention is not particularly limited, and oral administration and intravenous administration can be selected according to the symptoms, but oral administration in which the drug undergoes intestinal absorption is preferably used. The dosage of the agent for preventing and improving liver dysfunction according to the present invention is appropriately determined according to age, weight, sex, symptom, sensitivity to drugs and the like. Usually, it is administered in a dose range of 10 to 1000 mg / kg / day, preferably in a dose range of 50 to 500 mg / kg / day, in a single dose or divided into multiple doses per day. The dosage may be adjusted according to the situation of improvement.

  By adding the ozonized oil and fat having a liver dysfunction prevention / improvement action to a food or drink or a feed, it can be made into a food or drink for preventing or improving a liver dysfunction or a feed for preventing or improving a liver dysfunction. In addition, the food and drink for preventing and improving liver dysfunction according to the present invention and the diet for preventing and improving liver dysfunction are effective in suppressing the accumulation of liver fat during obesity, and the prevention and improvement of symptoms of fatty liver and metabolic syndrome. Can be used for In addition, the food and drink for preventing and improving liver dysfunction according to the present invention and the feed for preventing and improving liver dysfunction are for reducing or suppressing the concentration of PAI-1 in the blood or for reducing the blood AST or ALT concentration. Can be used. Foods and drinks include edible fats and oils such as butter, margarine, shortening, lard, seasonings such as soy sauce, sauce, mayonnaise, dressing, livestock meat, fish, vegetables, fruit processed foods, side dishes, dairy products, confectionery, ice confectionery All kinds of foods and beverages such as soft drinks such as alcoholic beverages, alcoholic beverages, milk beverages, fruit beverages and tea beverages can be used as general foods or health functional foods, and feeds such as pets, livestock, poultry, and cultured fish It can be used for any feeding application. These foods and drinks and feeds are labeled with general foods and drinks or feeds that have no liver dysfunction prevention / improvement action by indicating that they have a liver dysfunction prevention / improvement action in the manufacturing process or distribution process. Can be distinguished.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.

  The liver function of the ozonized oil and fat and the physiological effects of metabolic syndrome on pathogenesis were confirmed by animal experiments using obese model rats as test animals.

  The test animals include Zucker (fa / fa) rats that are homozygotes for the leptin receptor recessive mutant gene fa and 5-week-old males of wild-type dominant gene homozygotes Zucker (+ / +) rats. Individuals were used. Zucker (fa / fa) rat is a model that shows overeating, obesity, hyperlipidemia, and fatty liver, because the leptin receptor gene involved in feeding and energy metabolism is impaired by the leptin transmission pathway. Is an animal. 5-week-old male Zucker (fa / fa) rats and 5-week-old male Zucker (+ / +) rats were purchased from SLC Japan.

  The ozonized fats and oils were given to the test animals by adding and mixing ozonized olive oil obtained by ozonizing olive oil to the diet. Ozonized olive oil was prepared by dissolving 120 g of olive oil (manufactured by Wako Pure Chemical Industries, Ltd.) in 3 volumes of dichloromethane, cooling to 0 ° C. and stirring, while generating ozone generator ED-OG-R6 (Ecodesign Corporation). Made by supplying oxygen gas at a rate of 5 L / min and venting 2.5 g of ozone gas for 2.5 hours (the molecular weight of olive oil is 885.4, and the oleic acid content in olive oil is 80%) The number of double bonds in the molecule was 3, and the ozonization reaction rate was about 64%). The partial structure of the obtained ozonide was confirmed by NMR. After ozonization, the contained dichloromethane was removed by vacuum drying to obtain an ozonized olive oil sample. The diet was prepared with the composition shown in Table 1 according to the AIN-76 rodent standard feed composition by the National Institute of Nutrition. Control diets (Normal and Control) fed to Zucker (+ / +) and Zucker (fa / fa) rats, respectively, include diet containing 6.5% corn oil and 0.5% olive oil rich in oleic acid. As an experimental food to be fed to Zucker (fa / fa), as for the low ozonized oil and fat administration group (Low Ozone group; LO group), corn oil 6.5%, olive oil 0.45% and ozonized olive oil A diet containing 0.05% was used, and a diet containing 6.5% corn oil and 0.5% ozonized olive oil was used for the high ozonized oil / fat administration group (High ozone group; HO group).

  Obese Zucker (fa / fa) rats and wild-type Zucker (+ / +) rats are room temperature 23 ± 2 ° C., light period 12 hours (7-19 o'clock), dark period 12 hours (19-next 7 o'clock) ) Were kept in the animal room under the light conditions by feeding from 19:00 to next 9 o'clock. First, Zucker (fa / fa) rats and Zucker (+ / +) rats were each fed a control diet and preliminarily raised for 1 week. Divided into 3 groups, oil-fat administration group and high ozonized oil-fat administration group, each diet was fed and paired for 4 weeks. For Zucker (+ / +) rats, the control diet was fed as a positive control. Continued for 4 weeks. The body weight and food intake of each rat were measured every day during the breeding period, and the final weight after fasting for 9 hours on the final breeding day was measured. The results are shown in Table 2.

  After measuring the final body weight, each rat was anesthetized with diethyl ether and sacrificed by abdominal aorta blood sampling to remove blood, liver, and adipose tissue. The removed liver and adipose tissue were weighed. The results are shown in Table 2. The blood was allowed to stand and then centrifuged (3000 rpm, 15 minutes, 4 ° C.) to obtain serum. The liver, adipose tissue, and serum of the test rat obtained here were subjected to the following measurement test.

  As a result of comparing the body weight, food intake, white adipose tissue weight, and liver weight of each rat, the body weight, food intake, liver weight, and white adipose tissue weight of obese rats were significantly increased compared to wild type rats. There was no significant difference in feeding efficiency. Moreover, there was no difference in the final body weight, food intake, and white adipose tissue weight among the groups of obese rats, but the liver weight decreased by 7% in the LO group due to the consumption of ozonized oils and fats. In the HO group, a significant decrease of 19% was observed.

[Measurement of liver lipid concentration]
In order to measure the total amount of triglycerides, cholesterol and phospholipids contained in the liver of the obtained test rats, the total lipid components of the liver extracted from the rats were analyzed by the method of Folch et al. (Folch J et al., J. Biol Chem., 226, 497-509, 1957), a liver lipid extract was prepared by extraction and concentration by the following procedure. 0.1 g of the extracted liver was homogenized with 7.5 mL of methanol and 15 mL of chloroform, and then heated at 37 ° C. for 30 minutes, and a chloroform / methanol mixture (chloroform: methanol = 2: 1, v / v) was added. And the mixed solution made to 25 mL was filtered. Subsequently, about 20% of distilled water was added to the filtrate, mixed by inversion, and left overnight in a low temperature room (4 ° C.). The chloroform layer separated after standing was concentrated under reduced pressure using an evaporator under argon gas, and re-dissolved in petroleum ether to make a total volume of 25 mL as a liver lipid extract.

[Liver triglyceride concentration]
The liver triglyceride concentration was quantified by the method of Fletcher et al. (Fletcher MJ. Et al., Clin. Chim. Acta., 22, 393-397, 1968). 0.5 mL for Zucker (fa / fa) rats and 2 mL of liver lipid extract for Zucker (+ / +) rats were dried, and 5 mL chloroform and 1.0 g silica gel (Mallinckrodt Baker) were added. Then, after shaking for 5 minutes with a shaker, centrifugation (3000 rpm, 10 minutes, 10 ° C.) was performed. After 1 mL of the supernatant was collected and dried, 2 mL of an isopropanol / distilled water mixture (isopropanol: H ₂ O = 9: 1, v / v) and 0.6 mL of a 5% KOH solution (in isopropanol: H ₂₎ After adding O = 2: 3) and stirring, the mixture was heated in a water bath at 60 to 70 ° C. for 30 minutes for saponification. After returning to room temperature, 1 mL of 0.003 mol sodium metaperiodate and 0.5 mL of acetylacetone solution were added and stirred, and heated in a 50 ° C. water bath for 30 minutes for color development. After returning to room temperature again, the absorbance at a wavelength of 405 nm was measured. The results are shown in Table 3.

[Hepatic cholesterol concentration]
Regarding the liver cholesterol concentration, cholesterol E-test Wako (manufactured by Wako Pure Chemical Industries, Ltd.) was used, and cholesterol oxidase DAOS method (Allain CC et al., Clin. Chim. Acta., 20, 470-475, 1974 ). 1 mL of liver lipid extract was dried, 10 μL of 10% triton (in isopropanol) was added and stirred, followed by addition of 1.5 mL of color reagent and stirring. Then, warm at 37 ° C. for 5 minutes, add 1.5 mL of chloroform, shake with a shaker for 5 minutes, perform centrifugation (3000 rpm, 20 minutes, 10 ° C.), and the supernatant at a wavelength of 600 nm. Absorbance was measured. The results are shown in Table 3.

[Liver phospholipids]
The liver phospholipid concentration was quantified by the method of Rouser et al. (Fletcher MJ. Et al., Clin. Chim. Acta., 22, 393-397, 1968). After 0.5 mL of the liver lipid extract was dried and 1 mL of 70% perchloric acid was added, pyrolysis was performed at 180 to 190 ° C. for 30 to 60 minutes until the solution became colorless. Subsequently, the liquid temperature was returned to room temperature, 5 mL of distilled water, 1 mL of 2.5% ammonium molybdate solution and 1 mL of 10% ascorbic acid aqueous solution were added and stirred, and the mixture was stirred in a boiling bath at 100 ° C. for 5 minutes. Colored by heating. After returning to room temperature again, the absorbance at a wavelength of 820 nm was measured. The results are shown in Table 3.

  As a result of measuring the total amount of triglyceride, cholesterol and phospholipid contained in the liver of the test rat, the triglyceride concentration per gram of liver was significantly increased in the obese type compared to the wild type, and the phospholipid concentration A significant decrease was observed for. With the intake of ozonized fats and oils, the triglyceride concentration was significantly decreased by 47% in the HO group, and the phospholipid concentration was significantly increased by 25% in the HO group. In the whole liver, significant accumulation was observed in the obese type compared to the wild type for all of the triglyceride, cholesterol and phospholipid concentrations. Ingestion of ozonized fats and oils showed a remarkable suppression of 57% in the HO group for the accumulation of triglycerides.

[Measurement of serum lipid concentration and glucose concentration]
Triglyceride, cholesterol, phospholipid, free fatty acid, and glucose concentration contained in the serum of the obtained test rat were measured.

[Serum triglyceride]
The serum glyceride concentration was quantified by measuring the absorbance at a wavelength of 600 nm by triglyceride E-Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.) by the GPO / DAOS method.

[Serum cholesterol]
The serum cholesterol concentration was quantified by measuring the absorbance at a wavelength of 600 nm by cholesterol oxidase / DAOS method using cholesterol E-Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.). The results are shown in Table 4.

[Serum phospholipids]
The serum phospholipid concentration was quantified by measuring the absorbance at a wavelength of 600 nm by the choline oxidase DAOS method using Phospholipid C-Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.). The results are shown in Table 4.

[Serum free fatty acids]
The serum free fatty acid concentration was quantified by measuring the absorbance at a wavelength of 550 nm by the ACS / ACOD method using NEFA C-Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.). The results are shown in Table 4.

[Serum glucose]
The serum glucose concentration was quantified by measuring the absorbance at a wavelength of 505 nm using a glucose CII-Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.) by the mutarose / GOD method. The results are shown in Table 4.

  As a result of measuring triglyceride, cholesterol, phospholipid, free fatty acid and glucose contained in the serum of the test rat, the triglyceride concentration, cholesterol concentration, phospholipid concentration and free fatty acid concentration were significant in the obese type compared to the wild type. An increase was observed. By eating ozonized fats and oils, the triglyceride concentration increased significantly in the HO group, the free fatty acid concentration increased by 13% in the LO group, the significant increase by 32% in the HO group, and the cholesterol concentration decreased by LO. There was a 14% decrease trend in the group and a significant decrease of 18% in the HO group. Regarding the glucose concentration, no difference was observed between the groups.

[Measurement of serum protein concentration]
Serum plasminogen activation inhibitor (PAI-1), serum C-reactive protein (CRP), serum insulin, serum adiponectin, and serum monocyte chemotaxis contained in the serum of the obtained test rats The concentration of active factor 1 (monocyte chemoattractant protein-1; MCP-1) was measured.

[Serum plasminogen activation inhibitor (PAI-1)]
Serum PAI-1 concentration was measured by ELISA using IMUCLONE ^™ Rat PAI-1 ELISA (manufactured by American Diagnostica Inc.). The results are shown in Table 5.

[Serum C-reactive protein (CRP)]
Serum CRP concentration was measured by ELISA using C-Reactive Protein ELISA Kit (Alpha Diagnostic International Inc.). The results are shown in Table 5.

[Serum insulin]
Serum insulin concentration was measured by ELISA using Levis Insulin-Rat T (manufactured by Shiba Goat Co., Ltd.). The results are shown in Table 5.

[Serum adiponectin]
Serum adiponectin concentration was measured by ELISA using a mouse / rat adiponectin ELISA kit (manufactured by Otsuka Pharmaceutical Co., Ltd.). The results are shown in Table 5.

[Serum monocyte chemotactic factor 1 (MCP-1)]
Serum MCP-1 concentration was measured by ELISA using MCP-1 Rat ELISA Kit (Invitrogen). The results are shown in Table 5.

  As a result of measuring the concentrations of PAI-1, CRP, insulin, adiponectin, and MCP-1 contained in the serum of the test rat, a significant increase was observed in the obese type as compared to the wild type. Due to the consumption of ozonized oils and fats, 27% in the LO group and 38% in the HO group were shown to decrease. There was no difference between the groups regarding CRP concentration, insulin concentration and adiponectin concentration. Regarding the serum MCP-1 concentration, a marked increase was observed in the HO group due to the consumption of ozonized fats and oils.

[Measurement of liver dysfunction index enzyme activity]
The liver function of the obtained test rats was analyzed by measuring aspartate aminotransferase (AST) activity and alanine aminotransferase (ALT) activity as liver dysfunction indicator enzymes.

[Aspartate aminotransferase (AST)]
About AST activity, the light absorbency in wavelength 555nm was measured and quantified by POP * TOOS method using transaminase CII-Test Wako (made by Wako Pure Chemical Industries Ltd.). The results are shown in Table 6.

[Alanine aminotransferase (ALT)]
About ALT activity, the light absorbency in wavelength 555nm was measured and quantified by POP * TOOS method using transaminase CII-Test Wako (made by Wako Pure Chemical Industries Ltd.). The results are shown in Table 6.

  As a result of measuring the AST activity and the ALT activity of the test rat, both the AST activity and the ALT activity were significantly increased in the obese type compared to the wild type. As a result of eating ozonized fats and oils, the AST activity showed a 6% decrease in the LO group, a 29% significant decrease in the HO group, and a 33% decrease in the HO activity in the HO group.

[Measurement of liver triglyceride metabolism-related enzyme activity]
The liver triglyceride metabolism-related enzyme activity in the liver of the obtained test rat was measured. Examples of liver triglyceride metabolism-related enzymes include fatty acid synthase (FAS), carnitine palmitoyltransferase (CPT), peroxisomal β-oxidation-related enzymes, phosphatidate phosphohydrolase; PAP) was measured, and a liver homogenate excised from the rat was prepared as a measurement sample by the following procedure. 2 g of the extracted liver was cooled with 10 mM Tris-HCl buffer (pH 7.4) containing 6 volumes of 0.25 M sucrose (Wako Pure Chemical Industries, Ltd.) and 1 mM EDTA (Katayama Chemical Co., Ltd.). After homogenization, centrifugation (3000 rpm, 700 × g, 10 minutes, 4 ° C.) was performed. Subsequently, the supernatant was centrifuged (12000 rpm, 10,000 × g, 10 minutes, 4 ° C.) to precipitate the mitochondrial fraction. Further, this supernatant was centrifuged (40000 rpm, 125000 × g, 60 minutes, 4 ° C.) to obtain a cytoplasmic fraction of the supernatant and a precipitated microsomal fraction.

[Fatty Acid Synthase (FAS)]
The fatty acid synthase activity was measured by the method of Kelley et al. (Kelley DS et al., Biochem. J., 235, 87-90, 1986). 100 mM potassium phosphate buffer (pH 7.0) containing acetyl CoA (manufactured by Sigma-Aldrich) 0.05 mM, NADPH (manufactured by Oriental Yeast Co., Ltd.) 0.3 mM, EDTA (manufactured by Katayama Chemical Co., Ltd.) 0.2 mM The cytosolic fraction was added to the sample, and the absorbance at a wavelength of 340 nm was measured at 30 ° C. for 2 minutes to determine a decrease in NADPH absorbance in the blank. Thereafter, malonyl CoA (manufactured by Sigma-Aldrich) was added and mixed so that the final concentration was 0.2 mM, the absorbance at a wavelength of 340 nm was traced for 3 minutes, and obtained according to the following formula when malonyl CoA was added. The decrease rate of NADPH was determined by subtracting the decrease in absorbance of the blank from the decrease in absorbance during the reaction, and the fatty acid synthase activity was calculated. The results are shown in Table 7.
Fatty acid synthase activity (nmol / min / mg protein) = {(decrease in absorbance during reaction−decrease in absorbance of blank) / 6220} × 10 ⁹ × (reaction volume mL / 1000) × (1 / measurement time min ) × (1 / protein amount mg)
6220 is the molar extinction coefficient of NADPH.

[Carnitine Palmitoyltransferase (CPT)]
Carnitine palmitoyltransferase activity was measured by the method of Markwell et al. (Markwell MA, et al., J. Biol. Chem., 248, 10, 3426-3432, 1973). Palmitoyl CoA (Funakoshi Co., Ltd.) 0.0375 mM, 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) (Tokyo Chemical Industry Co., Ltd.) 0.25 mM, EDTA (Katayama Chemical Co., Ltd.) 1 .25 mM, Triton X-100 (manufactured by Wako Pure Chemical Industries, Ltd.) A mitochondrial fraction was added to 58 mM Tris-HCl buffer (pH 8.0) containing 0.1%, and the absorbance at 412 nm was 5 at 27 ° C. The increase in reduced DTNB in the blank was determined by measuring for minutes. Thereafter, L-carnitine (manufactured by Sigma-Aldrich) was added so that the final concentration was 1.25 mM, and the absorbance at a wavelength of 412 nm in a total reaction volume of 1 mL was traced for 3 minutes. -The production rate of CoA was determined by subtracting the increase in absorbance of the blank from the increase in absorbance at the time of reaction obtained when carnitine was added, and the carnitine palmitoyltransferase activity was calculated. The results are shown in Table 7.
Carnitine palmitoyltransferase activity (nmol / min / mg protein) = {(Increase in absorbance during reaction−Increase in absorbance of blank) / 13600} × 10 ⁹ × (reaction volume mL / 1000) × (1 / protein amount mg )
Note that 13600 is the molar extinction coefficient of CoA.

[Peroxisomal β-oxidation]
Peroxisome β oxidation activity was measured by the method of Lazarow (Lazarow PB, Methods Enzymol. Academic Press, New York, 72, 315-319, 1981). NAD (manufactured by Oriental Yeast Co., Ltd.) 0.2 mM, DTT (manufactured by Katayama Chemical Co., Ltd.) 1 mM, bovine serum albumin (manufactured by Sigma-Aldrich) 75 μg / mL, Triton X-100 (manufactured by Wako Pure Chemical Industries, Ltd.) 100 μL CoA (Sigma-Aldrich) 0.1 mM, FAD (Sigma-Aldrich) 0.01 mM, potassium cyanide (Katayama Chemical Co., Ltd.) 1 mM mitochondrial fraction in 47 mM Tris-HCl buffer (pH 8.0) Minutes were added, and the absorbance at a wavelength of 340 nm was measured at 37 ° C. for 3 minutes to determine the increase in NADH absorbance in the blank. Thereafter, 1 mM palmitoyl CoA (manufactured by Funakoshi Co., Ltd.) was mixed so that the final concentration was 0.01 mM, and the absorbance at a wavelength of 340 nm in a total reaction volume of 1 mL was traced for 5 minutes. By subtracting the increase in the absorbance of the blank from the increase in the absorbance during the reaction obtained at the time of addition, the production rate of NADH was determined, and the peroxisome β-oxidation activity was calculated. The results are shown in Table 7.
Peroxisome β-oxidation activity (nmol / min / mg protein) = {(Increase in absorbance during reaction−Increase in absorbance of blank) / 6220} × 10 ⁹ × (reaction volume mL / 1000) × (1 / measurement time min ) X (1 / protein amount mg)
6220 is the molar extinction coefficient of NADH.

[Phosphatidate phosphohydrolase (PAP)]
The phosphatidic acid phosphohydrolace activity was measured by the method of Walton et al. (Walton, PA et al., Anal. Biochem., 151, 479-486, 1985). Phosphatidylcholine (manufactured by Sigma-Aldrich) mixed by sonication using 0.9% sodium chloride 1.0 mM, EDTA (manufactured by Katayama Chemical Co., Ltd.) 50 mM Tris-HCl buffer (pH 7. The microsome fraction is added to each of the case where magnesium chloride (manufactured by Wako Pure Chemical Industries, Ltd.) is added to 0) so that the final concentration is 3.25 mM, and the total volume is 0.2 mL. And incubated at 37 ° C. for 15 minutes. Subsequently, the reaction solution contains 0.8 mL of color developing solution (0.1% sodium lauryl sulfate (manufactured by Wako Pure Chemical Industries, Ltd.), 1.25% ascorbic acid, and 0.32% ammonium molybdate tetrahydrate. .375M sulfuric acid aqueous solution) was added and incubated at 45 ° C. for 20 minutes to develop inorganic phosphorus as a reaction product, and the absorbance at a wavelength of 820 nm was measured. Measure the amount of phosphorus produced from microsomes by reaction without phosphatidic acid (Sigma-Aldrich) and phosphatidylcholine to determine the amount of inorganic phosphorus in the blank, and calculate the amount of inorganic phosphorus as the reaction product according to the following formula It was.
Inorganic phosphorus production (μg) = Phosphorus production by reaction (phosphorus production from microsomes + phosphorus production from phosphatidic acid and phosphatidylcholine)
The phosphatidic acid phosphohydrolace activity was calculated from the obtained inorganic phosphorus production amount according to the following formula. The results are shown in Table 7.
Phosphatidic acid phosphohydrolace activity (nmol / min / mg protein) = inorganic phosphorus production amount × 10 ³ × (1 / 3.97) × (1/15 min) × (1 / protein mg).
The atomic weight of phosphorus was 30.97.

  As a result of measurement of liver triglyceride metabolism-related enzyme activity in the liver of test rats, fatty acid synthase (FAS), peroxisome β-oxidation activity, phosphatidic acid phosphohydrolase activity were significantly increased in obese type compared to wild type Was recognized. No effect on the enzyme activity was observed by eating ozonized fats and oils.

[Measurement of lipid excretion rate]
Fecal lipid excretion rate was measured by the method of Ikeda et al. (Kunio Ikeda, “Function of Lipids”, Food Function Research Method, Koso Co., Ltd., 2000, P21-24). Feces were collected for 2 days immediately before sacrifice of each rat, lyophilized and ground. 0.25 g is precisely weighed and about 2 drops of concentrated hydrochloric acid (made by Wako Pure Chemical Industries, Ltd.) per 7 to 8 mL is added dropwise. A mixture of heptane, diethyl ether and purified ethanol (heptane: diethyl ether: purified ethanol = 1: 1: 1, 4 ml of v / v) was added, and the mixture was shaken with a shaker for 1 minute and centrifuged at 3000 rpm for 10 minutes. Collect a certain amount of the upper layer, add 4 mL of a mixed solution of heptane / diethyl ether / purified ethanol / deionized water (1: 1: 1: 1, v / v, upper layer only), shake with a shaker for 1 minute, and add 10 mL at 3000 rpm. Centrifuged for minutes. Subsequently, this operation was repeated once again, and after collecting the upper layer, it was dried and weighed. The results are shown in Table 8.

  As a result of the measurement, no significant difference was observed between the wild-type and obese-type groups in terms of fecal lipid excretion rate.

[Results of feeding ozonized fats and oils]
By feeding the ozonized oil of the present invention, the liver weight of the test rat was reduced, and accumulation of glycerides in the liver was suppressed. No effects on fatty acid synthesis / degradation enzyme activity were observed with ozonized fats and fats, but no change in fecal lipid excretion rate was observed, but serum triglyceride concentration was increased It was confirmed that ozonized oils and fats have an action of releasing glycerides from the liver into the blood. Contrary to this, blood cholesterol was significantly reduced, and it was confirmed that the action of ozonized oils and fats was selective only to triglycerides. As a result of measuring the concentrations of PAI-1, CRP and MCP-1 which are inflammatory factors contained in rat serum, PAI-1 and CRP showed a tendency to decrease and MCP-1 increased due to feeding. In addition, serum concentration of adiponectin, an anti-inflammatory factor, decreased. Each of these measurement results is tested by simple regression analysis, Tukey-Kramer method and Student's t test (WS Gosset, Biometrika, vol. 6, no. 1, p1-25, 1908), and the risk rate is less than 5%. Were analyzed as significant differences. As a result of simple regression analysis, the relationship between the suppression of triglyceride accumulation in the liver and the concentrations of CRP, MCP-1, and adiponectin is low, and PAI-1 shows a positive correlation with the suppression of triglyceride accumulation in the liver. It was found that only the concentration (FIG. 1). It was confirmed that the effect of preventing and improving the liver dysfunction of the ozonized fat of the present invention is through the suppression of PAI-1 concentration in blood.

Claims (4)

An agent for preventing or improving liver dysfunction caused by metabolic abnormalities, comprising ozonized olive oil as an active ingredient. Prevention improving agent according to claim 1 which is used to suppress an increase in the concentration of plasminogen activator inhibitor in the blood (PAI-1). The preventive / ameliorating agent according to claim 1 or 2, which is used as a health functional food. The prevention-improving agent according to claim 1 or 2, which is used as a feed additive.

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