Ho-1 expression enhancer and Anti-inflammatory agent

Abstract

To provide an agent capable of effectively enhancing the expression of HO-1, and / or an agent capable of effectively inhibiting inflammation inside the body.SOLUTION: The present invention provides an HO-1 expression enhancer or the like, comprising, as an active ingredient, an ozonized polyunsaturated fatty acid or an ester thereof, or fats or oils containing an ozonized polyunsaturated fatty acid as a constituent fatty acid.SELECTED DRAWING: Figure 5

Description

Technical Field

[0001] The present invention relates to an HO-1 expression enhancer and an anti-inflammatory agent.

Background Art

[0002] Inflammation is a biological defense reaction to remove the causative agent of injury and the damaged tissue when biological tissue is damaged due to bacterial infection or the action of physicochemical factors. However, excessive inflammation is known to cause various diseases, and there are cases where inflammation in the body should be suppressed.

[0003] HO-1 (Heme Oxygenase 1) is an important enzyme as a biological defense mechanism that protects cells from oxidative stress. Deficiency of HO-1 enhances cell injury caused by oxidative stress and promotes inflammation. On the other hand, enhanced expression of HO-1 suppresses oxidative stress and suppresses inflammation caused by cell injury. Therefore, in order to suppress inflammation in the body, the use of substances that enhance the expression of HO-1 is desired.

[0004] Examples of substances that enhance the expression of HO-1 include 10-oxo-trans-11-octadecenoic acid, 10-oxo-trans-11,cis-15-octadecadienoic acid, and 10-oxo-cis-6,trans-11-octadecadienoic acid (Patent Document 1), enzymatically synthesized glycogen or its α-amylase digest (Patent Document 2), etc. However, it has not been known what kind of influence ozonized polyunsaturated fatty acids and their esters have on the expression of HO-1.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Summary of the Invention

[0006] An object of the present invention is to provide an agent that can effectively enhance the expression of HO-1 and / or an agent that can effectively suppress inflammation in the body. [Means for Solving the Problems]

[0007] As a result of intensive studies by the present inventors, it has been found that ozonized polyunsaturated fatty acid esters and oils and fats containing ozonized polyunsaturated fatty acids as constituent fatty acids have an action of enhancing the mRNA expression of HO-1. In addition, it has been found that ozonized polyunsaturated fatty acid esters and oils and fats containing ozonized polyunsaturated fatty acids as constituent fatty acids have an action of suppressing the mRNA expression of inflammatory factor genes. The present invention has been completed based on these findings.

[0008] That is, the present invention is as specified by the following matters. [1] An agent for enhancing HO-1 expression, comprising an ozonized polyunsaturated fatty acid or its ester, or an oil or fat containing an ozonized polyunsaturated fatty acid as a constituent fatty acid as an active ingredient. [2] The agent for enhancing HO-1 expression according to [1] above, wherein the polyunsaturated fatty acid is linoleic acid or linolenic acid. [3] The agent for enhancing HO-1 expression according to [1] or [2] above, wherein the oil or fat is olive oil. [4] An anti-inflammatory agent that suppresses inflammation in the body by enhancing the expression of HO-1, comprising an ozonized polyunsaturated fatty acid or its ester, or an oil or fat containing an ozonized polyunsaturated fatty acid as a constituent fatty acid as an active ingredient. [5] An anti-inflammatory agent for inflammation in the body, comprising an ozonized polyunsaturated fatty acid or its ester, or an oil or fat containing an ozonized polyunsaturated fatty acid as a constituent fatty acid as an active ingredient. [6] The anti-inflammatory agent according to [4] or [5] above, wherein the polyunsaturated fatty acid is linoleic acid or linolenic acid. [7] An anti-inflammatory agent according to any one of [4] to [6] above, wherein the fat or oil is olive oil.

[0009] Further, as another embodiment of the present invention, for example, the following can be mentioned. [1-1] Use of an ozonized polyunsaturated fatty acid or its ester, or a fat or oil containing an ozonized polyunsaturated fatty acid as a constituent fatty acid, for producing an HO-1 expression enhancer. [1-2] An ozonized polyunsaturated fatty acid or its ester, or a fat or oil containing an ozonized polyunsaturated fatty acid as a constituent fatty acid, for use in enhancing HO-1 expression. [1-3] A method for enhancing HO-1 expression, comprising administering an effective amount of an ozonized polyunsaturated fatty acid or its ester, or a fat or oil containing an ozonized polyunsaturated fatty acid as a constituent fatty acid, to a subject in need thereof. [4-1] Use of an ozonized polyunsaturated fatty acid or its ester, or a fat or oil containing an ozonized polyunsaturated fatty acid as a constituent fatty acid, for producing an anti-inflammatory agent that suppresses inflammation in the body by enhancing the expression of HO-1. [4-2] An ozonized polyunsaturated fatty acid or its ester, or a fat or oil containing an ozonized polyunsaturated fatty acid as a constituent fatty acid, for use in anti-inflammatory treatment that suppresses inflammation in the body by enhancing the expression of HO-1. [4-3] An anti-inflammatory method for suppressing inflammation in the body by enhancing the expression of HO-1, comprising administering an effective amount of an ozonized polyunsaturated fatty acid or its ester, or a fat or oil containing an ozonized polyunsaturated fatty acid as a constituent fatty acid, to a subject in need thereof. [5-1] Use of an ozonized polyunsaturated fatty acid or its ester, or a fat or oil containing an ozonized polyunsaturated fatty acid as a constituent fatty acid, for producing an anti-inflammatory agent against inflammation in the body. [5-2] An ozonized polyunsaturated fatty acid or its ester, or a fat or oil containing an ozonized polyunsaturated fatty acid as a constituent fatty acid, for use in anti-inflammatory treatment against inflammation in the body. [5-3]An anti-inflammatory method for inflammation in the body, which includes administering an effective amount of ozonized polyunsaturated fatty acid or its ester, or an oil or fat having ozonized polyunsaturated fatty acid as a constituent fatty acid, to a subject in need thereof.

Advantages of the Invention

[0010] According to the present invention, the expression of HO-1 can be effectively enhanced, and / or the inflammation in the body can be effectively suppressed.

Brief Description of the Drawings

[0011]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Modes for Carrying Out the Invention

[0012] The present invention relates to a "HO-1 expression enhancer", an "anti-inflammatory agent that suppresses inflammation in the body by enhancing the expression of HO-1", and an "anti-inflammatory agent for inflammation in the body". All of these agents contain any one or two or more of the following (1) to (3) as active ingredients. (1) Ozonized polyunsaturated fatty acid (2) Ozonized polyunsaturated fatty acid ester (3) Oil or fat having ozonized polyunsaturated fatty acid as a constituent fatty acid

[0013] In the present invention, the ozonized polyunsaturated fatty acid means a polyunsaturated fatty acid that has been subjected to an ozonization treatment. By the ozonization treatment, the double bonds of the unsaturated fatty acid are ozonized to generate trioxolane. The method of the ozonization treatment is not particularly limited and can be carried out by a known method. For example, it can be carried out by bubbling (conducting) ozone gas through the object.

[0014] In the present invention, the polyunsaturated fatty acid is not particularly limited as long as it is an unsaturated fatty acid having two or more double bonds. In the present invention, examples of the polyunsaturated fatty acid include linoleic acid, linolenic acid, arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid, etc. Linoleic acid or linolenic acid is preferable, and linolenic acid is more preferable. Examples of linolenic acid include α-linolenic acid and γ-linolenic acid having different double bond positions, and either can be used, but α-linolenic acid is more preferable.

[0015] In the present invention, the polyunsaturated fatty acid ester is not particularly limited as long as it is an esterified product of the aforementioned polyunsaturated fatty acid. In the present invention, examples of the polyunsaturated fatty acid ester include polyunsaturated fatty acid alkyl esters, and polyunsaturated fatty acid lower alkyl esters are preferable, polyunsaturated fatty acid methyl esters or polyunsaturated fatty acid ethyl esters are more preferable, and polyunsaturated fatty acid ethyl esters are even more preferable. As the polyunsaturated fatty acid ethyl ester, ethyl linoleate or ethyl linolenate is preferable, ethyl linolenate is more preferable, and ethyl α-linolenate is even more preferable.

[0016] In the present invention, the oil or fat containing polyunsaturated fatty acids as constituent fatty acids is not particularly limited as long as it contains one or more of the aforementioned polyunsaturated fatty acids as constituent fatty acids. In the present invention, the oil or fat may be any one of monoglyceride, diglyceride, or triglyceride, or a mixture thereof. In the present invention, examples of the oil or fat include oils or fats in which 1% or more, preferably 3% or more, more preferably 5% or more of the constituent fatty acids are polyunsaturated fatty acids. For example, it may be vegetable oil, animal oil, or processed oil derived therefrom. Specifically, examples of vegetable oil include olive oil, rapeseed oil, corn oil, soybean oil, cottonseed oil, safflower oil, sesame oil, peanut oil, sunflower oil, grape seed oil, palm oil, coconut oil, rice oil, etc., and examples of animal oil include beef tallow, lard, milk fat, fish oil, etc. It may be a hardened oil or transesterified oil of these oils, a liquid oil or solid fat obtained by fractionating these oils, etc. Further, two or more of these oils may be used in combination. Preferably, olive oil, rapeseed oil, safflower oil, sunflower oil, etc. can be mentioned, and olive oil is more preferable.

[0017] Fatty acid esters or oils and fats are known to be metabolized in vivo and decomposed into fatty acids before being absorbed. Therefore, in the present invention, when the aforementioned (2) ozonized polyunsaturated fatty acid ester or (3) oil or fat containing ozonized polyunsaturated fatty acids as constituent fatty acids is administered to an animal, it can be considered that it is metabolized in the animal's body and decomposed into (1) ozonized polyunsaturated fatty acids and acts in the animal's body. Therefore, in the present invention, (1) ozonized polyunsaturated fatty acids, (2) ozonized polyunsaturated fatty acid esters, and (3) oils and fats containing ozonized polyunsaturated fatty acids as constituent fatty acids can be considered to produce the same effects derived from the ozonized polyunsaturated fatty acids contained therein.

[0018] In the present invention, ozonized polyunsaturated fatty acids or their esters, or oils and fats containing ozonized polyunsaturated fatty acids as constituent fatty acids are used as active ingredients of a "HO-1 expression enhancer", an "anti-inflammatory agent that suppresses inflammation in the body by enhancing the expression of HO-1", and an "anti-inflammatory agent against inflammation in the body".

[0019] In the present invention, "used as an active ingredient" means containing an effective amount. The effective amount means an amount sufficient to exhibit a "HO-1 expression enhancing effect", an "anti-inflammatory effect of suppressing inflammation in the body by enhancing the expression of HO-1", and / or an "anti-inflammatory effect against inflammation in the body". Further, in the present invention, "used as an active ingredient" also means that other components may be contained as long as the effects of the present invention are not impaired. The effective amount may vary depending on the state of the administration subject (e.g., the degree of inflammation), the animal species, age (in months), sex, or body weight of the administration subject individual, etc. In the present invention, the effective amount is, for example, 10 to 3000 mg / kg / day, preferably 50 to 500 mg / kg / day, more preferably 100 to 300 mg / kg / day, and may be administered once a day or divided into multiple times a day.

[0020] In the present invention, "enhanced HO-1 expression" means enhancing the expression of HO-1. "Enhanced HO-1 expression" can be evaluated, for example, using the mRNA expression level and / or protein expression level of HO-1 as indicators. Known methods can be used for those evaluations.

[0021] HO-1 is an important enzyme as a biological defense mechanism that protects cells from oxidative stress. Deficiency of HO-1 enhances cell damage caused by oxidative stress and promotes inflammation. On the other hand, enhanced expression of HO-1 suppresses oxidative stress and suppresses inflammation caused by cell damage. Therefore, by enhancing the expression of HO-1, inflammation in the body can be suppressed (anti-inflammation).

[0022] In the present invention, "anti-inflammatory" means suppressing inflammation in the body. "Anti-inflammatory" can be evaluated, for example, using as indicators the amount of nitric oxide (NO) production, the mRNA expression level and / or protein expression level of oxidative stress response genes, and / or the mRNA expression level and / or protein expression level of inflammatory factor genes.

[0023] In the inflammatory reaction in the body, it is known that NO produced by macrophages acts as a mediator. While NO acts as a biological defense factor, when produced in large amounts during inflammation, it exacerbates inflammation. Therefore, by suppressing excessive NO production by macrophages, inflammation in the body (anti-inflammation) can be suppressed. In those evaluations, known methods can be used.

[0024] Examples of oxidative stress response genes include, for example, HO-1 and NQO-1 (NAD(P)H Quinone Oxidoreductase 1). Examples of inflammatory factor genes include, for example, MCP1 (Monocyte Chemotactic Protein1), TNFα (Tumor Necrosis Factor α), PTGS2 (Prostaglandin-Endoperoxide Synthase 2), IL1β (Interleukin 1 Beta), and IL6 (Interleukin 6). In those evaluations, known methods can be used.

[0025] In the present invention, ozonized polyunsaturated fatty acids or their esters, or oils and fats containing ozonized polyunsaturated fatty acids as constituent fatty acids can also be used as active ingredients of a "NO production inhibitor", "oxidative stress inhibitor", "antioxidant", "NQO-1 expression enhancer", "MCP1 expression inhibitor", "TNFα expression inhibitor", "PTGS2 expression inhibitor", "IL1β expression inhibitor", and / or "IL6 expression inhibitor".

[0026] In the present invention, "inflammation in the body" means inflammation occurring in the living body of an animal, excluding skin inflammation (dermatitis). The inflammation may be any inflammation of cells (including cultured cells), tissues, organs, or individuals. Further, the inflammation may be either chronic inflammation or acute inflammation. The site of occurrence, cause, degree, etc. of the inflammation are not particularly limited, and examples include myositis, arthritis, retinitis, gastritis, hepatitis, bronchitis, esophagitis, enteritis, pancreatitis, colitis, nephritis, thyroiditis, etc., but one or more of these may be excluded. By suppressing inflammation with the anti-inflammatory agent of the present invention, it is also expected to prevent and / or treat pathological conditions and diseases caused by inflammation. The pathological conditions and diseases caused by inflammation are not particularly limited, and examples include cell damage due to chronic inflammation, rheumatism, atherosclerosis, multiple sclerosis, asthma, allergic diseases, neuroinflammatory diseases, type 2 diabetes, hyperglycemia, insulin resistance, hypertension, NAFLD (non-alcoholic fatty liver disease), fatty liver, liver diseases, liver dysfunction, dyslipidemia, Alzheimer's disease, dementia, asthma, heart diseases, cancer, etc., but one or more of these may be excluded.

[0027] The "HO-1 expression enhancer", "anti-inflammatory agent that suppresses inflammation in the body by enhancing the expression of HO-1", and "anti-inflammatory agent against inflammation in the body" of the present invention can be used, for example, as pharmaceuticals, food and beverages, etc., or in combination therewith.

[0028] The method of providing the product as a pharmaceutical is not particularly limited. For example, the active ingredient can be used as it is, or formulated into various pharmaceutical dosage forms for oral or parenteral systemic or local administration by various known methods using pharmaceutically acceptable and appropriately selected additives according to the dosage form (such as carriers, excipients, diluents, binders, lubricants, disintegrants or disintegration aids, solubilizers, stabilizers, preservatives, antiseptics, bulking agents, thickeners, emulsifiers, dispersants, suspending agents, buffers, etc.). When the pharmaceutical is administered orally, it can be formulated into tablets (including sugar-coated tablets), capsules, granules, powders, pills, oral solutions, suspensions, emulsions, syrups, etc., or made into a dry product that is redissolved when used. When the pharmaceutical is administered parenterally, it can be formulated into injections (such as subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections), drip infusions, suppositories (such as rectal suppositories, vaginal suppositories), etc. In the case of injectable preparations, they can be provided in the form of unit-dose ampoules or multi-dose containers.

[0029] The method of providing the product as a food or beverage is not particularly limited. For example, the active ingredient can be provided as it is, or in solid, liquid, semi-liquid, granular, particulate, powdered, capsule, cream, or paste form, etc., together with other ingredients according to the form of the food or beverage. Here, the other ingredients are not particularly limited, and examples include various proteins, saccharides, fats, trace elements, vitamins, organic acid salts such as citric acid and acetic acid, etc. In addition, commonly used additives such as sweeteners like aspartame and stevia, acidulants like citric acid, malic acid, and tartaric acid, excipients like dextrin and starch, as well as colorants, flavors, bittering agents, buffers, thickening stabilizers, gelling agents, stabilizers, gum bases, binders, diluents, emulsifiers, dispersants, suspending agents, antioxidants, preservatives, antiseptics, fungicides, color developers, bleaching agents, brighteners, enzymes, seasonings, spice extracts, etc. can be added as appropriate.

[0030] Food and drink products include health foods, functional foods, foods for specified health use, dietary supplements, and foods for patients. The above-mentioned health foods, etc. may have the form of ordinary foods, but may also have the form of supplements (tablets, granules, fine granules, tablets, chewable tablets, capsules (soft capsules, hard capsules), etc.).

[0031] The specific forms of food and drink products are not particularly limited. For example, beverages such as soft drinks, carbonated drinks, nutritional drinks, fruit drinks, and milk drinks (including concentrated stock solutions and powder for adjustment of these beverages); carbohydrate-containing foods and drinks such as rice, noodles, breads, and pastas; Western confectioneries such as cookies and cakes, Japanese confectioneries such as steamed buns and yokan, various confectioneries such as candies, gums, yogurt, pudding, jelly, and frozen confections and ice confections; processed fishery and livestock products such as kamaboko, chikuwa, hamburg, ham, and sausage; dairy products such as processed milk, fermented milk, yogurt, butter, and cheese; processed oil and fat foods such as margarine, mayonnaise, shortening, whipped cream, and dressing; seasonings such as sauces and tare, etc. can be mentioned.

[0032] The administration targets of the "HO-1 expression enhancer", the "anti-inflammatory agent that suppresses inflammation in the body by enhancing the expression of HO-1", and the "anti-inflammatory agent against inflammation in the body" of the present invention are not particularly limited. For example, mammals such as humans, mice, rats, dogs, cats, rabbits, cows, horses, pigs, sheep, and goats, avian animals such as chickens and parrots, and fish such as carp, goldfish, and other cultured fish can be mentioned. In addition, the administration target of the "HO-1 expression enhancer" of the present invention may be, for example, a target that requires enhanced expression of HO-1, and the administration target of the "anti-inflammatory agent that suppresses inflammation in the body by enhancing the expression of HO-1" of the present invention may be, for example, a target that requires an anti-inflammatory agent that suppresses inflammation in the body by enhancing the expression of HO-1, and the administration target of the "anti-inflammatory agent against inflammation in the body" of the present invention may be, for example, a target that requires an anti-inflammatory agent against inflammation in the body.

[0033] Hereinafter, the present invention will be described more specifically using examples. However, the technical scope of the present invention is not limited to these examples.

Example

[0034] 1. Examination of the anti-inflammatory effect of ozonized oil The effect of oral ingestion of ozonized oil was examined using mice.

[0035] 1-1. Preparation of samples As the oil, olive oil (manufactured by Fujifilm Wako Pure Chemical Corporation) was used. The fractionation of triglyceride (TG) and unsaponifiable matter (US) in olive oil was carried out according to the following procedure. Olive oil dissolved in hexane was stirred with activated carbon to remove impurities. Olive oil was loaded onto a column filled with 20 times the amount of silica gel, and olive oil TG was obtained by eluting with hexane:2-propanol = 100:4. In addition, 25 g of olive oil was saponified with 1 M KOH / EtOH, and US in olive oil was extracted with diethyl ether to obtain olive oil US. The recovery rate at this time was 330 mg / 25 g of olive oil. 125 mL of dichloromethane (ultra-dehydrated) was added to olive oil TG or olive oil US, and it was cooled to -20 °C with acetone mixed with liquid nitrogen. Ozone gas generated from an ozone gas generator (ED-OG-R6, Eco Design Co., Ltd.) was bubbled into the reaction solvent at 1.0 L / min for 5 minutes. The ozone generation amount was set to 4 g / hr. Corn oil (manufactured by Fujifilm Wako Pure Chemical Corporation) was added to the obtained ozonized olive oil TG or ozonized olive oil US so as to achieve the ratio shown in Table 1, and the one from which dichloromethane was removed by an evaporator was used as a feed component.

[0036] 1-2. Feeding mice As test animals, 6-week-old male individuals of normal model C57BL / 6J mice (purchased from CLEA Japan, Inc.) and obese model db / db mice lacking the leptin receptor (purchased from CLEA Japan, Inc.) were used. The mice were divided into the following groups (n = 3 for the NO group; n = 6 for the CO group, zTG group, and zUS group). · NO group: C57BL / 6J mice were given 6.5% corn oil + 0.5% olive oil (control diet). · CO group: db / db mice were given 6.5% corn oil + 0.5% olive oil (control diet). · zTG group: db / db mice were given 6.5% corn oil + 0.5% ozonated olive oil TG. · zUS group: db / db mice were given 6.5% corn oil + 0.5% olive oil TG + 0.0058% ozonated olive oil US.

[0037] The feed composition was adjusted according to the AIN-76 rodent standard feed composition by the National Institute of Nutrition, USA (Table 1). Mice in each group were pre-fed with the control diet for 1 week, and then using Rodent CAFE (manufactured by Kirby Tea Oriental Co., Ltd.), each group was fed with the respective feed shown in Table 1 and bred in pairs of two for 4 weeks. Thereafter, the growth parameters were measured (Table 2). On the last day of breeding, after fasting for 9 hours, the mice were sacrificed by cardiac blood sampling under anesthesia, and the liver was excised for analysis.

[0038]

Table 1

[0039]

Table 2

[0040] 1-3. Evaluation of the mRNA expression levels of inflammatory factor genes mRNA was extracted from 0.1 g of liver using the RNeasy Lipid tissue Mini kit (QIAGEN). cDNA was prepared using Taqman Reverse Transcription reagents (Thermo Fisher Scientific). Real-time RT-PCR was performed using the ABI Prism 7000 real-time PCR sequence detection system (Thermo Fisher Scientific). TaqMan Universal PCR Master Mix (Thermo Fisher Scientific) was used for gene amplification. Assay-on-Demand, Gene Expression Products (Thermo Fisher Scientific) was used for gene quantification. As inflammatory factor genes, the mRNA expression levels of MCP1 (Taqman Assay ID: Mm004441242_m1) and TNFα (Taqman Assay ID: Mm00443258_m1) were evaluated. The data were normalized with the mRNA expression level of 18s RNA (Taqman Assay ID: Mm04277571_s1) as an internal standard.

[0041] The results are shown in Figure 1. In Figure 1, each data represents the mean ± standard deviation and is expressed as a relative expression level with the mean mRNA expression level of the CO group set to 100. The "*" in Figure 1 indicates a significant difference compared to the CO group (p < 0.05, Tukey’s HSD test). As is clear from Figure 1, the relative mRNA expression level of MCP1, an inflammatory factor, was significantly decreased in the zTG group compared to the CO group. Also, although not significant, the relative mRNA expression level of TNFα tended to decrease in the zTG group compared to the CO group. From this result, it was shown that ozonated TG in olive oil has an anti-inflammatory effect. Olive oil is known to have oleic acid, linoleic acid, and linolenic acid as constituent fatty acids. Therefore, it was considered that these ozonated fatty acids are the active components of the anti-inflammatory effect of ozonated TG.

[0042] 2. Examination of the anti-inflammatory effect of ozonated fatty acids Next, the anti-inflammatory effects of ozonated oleic acid, linoleic acid, or linolenic acid were examined.

[0043] 2-1. Preparation of ozonated fatty acid ethyl esters Ethyl oleate (>95.0%) was purchased from Tokyo Chemical Industry Co., Ltd., and ethyl linoleate (≥98%) and ethyl α-linolenate (≥99%) were purchased from Sigma Aldrich. Dichloromethane (ultra-dehydrated, 15 mL) was added to each fatty acid ethyl ester (150 mg), and the mixture was cooled to -20 °C with acetone mixed with liquid nitrogen. Ozone gas generated from an ozone gas generator (ED-OG-R6, Eco Design Co., Ltd.) was bubbled into the reaction solvent at 1.0 L / min for 5 minutes. The ozone generation amount was set to 4 g / hr. After bubbling, the mixture was stirred at -20 °C for 2 hours to stabilize the product. Dichloromethane was removed by an evaporator, and 15 mL of ethanol infinity pure (manufactured by Fujifilm Wako Pure Chemical Corporation) was added to prepare an ethanol solution of each ozonated fatty acid ethyl ester.

[0044] 2-2. Evaluation of NO production amount Mouse-derived macrophage cells RAW264 (purchased from RIKEN Cell Bank) were diluted with DMEM (Dulbecco's Modified Eagle Medium) to a concentration of 5.0×10 5 cells / mL, and 100 μL of the diluted cells were seeded in a 96-well plate and incubated at 5% CO 2 , 37 °C for 24 hours. Ethanol (control) and ethanol solutions of each fatty acid (oleic acid, linoleic acid, α-linolenic acid, ozonated oleic acid, ozonated linoleic acid, and ozonated α-linolenic acid) ethyl ester (each fatty acid ethyl ester approximately 1%) were mixed with 1 mL of DMEM containing 1% FBS (fetal bovine serum) to prepare the added samples. After removing the medium in each well with an aspirator, 100 μL of each added sample was added, and the plate was incubated at 5% CO 2 , 37 °C for 24 hours.

[0045] LPS (manufactured by Santa Cruz Biotechnology) derived from Escherichia coli 0111:B4 was diluted with PBS to prepare a 10 μg / mL solution (LPS stock), which was then further diluted 20-fold with DMEM containing 1% FBS to obtain a 500 ng / mL LPS solution. After adding 25 μL of the 500 ng / mL LPS solution (LPS(+)) or DMEM containing 1% FBS (LPS(-)) to each well, 5% CO 2 was incubated at 37 °C for 24 hours. 50 μL of the supernatant was taken from each well and transferred to a new 96-well plate. 50 μL of a 5% phosphoric acid aqueous solution containing 1% sulfanilamide was added, and the mixture was incubated at room temperature for 10 minutes. Then, 50 μL of a 0.1% NED (naphthylethylenediamine dihydrochloride) aqueous solution was added dropwise. After reacting at room temperature for 10 minutes, the absorbance at 540 nm was measured using an absorbance plate reader. A blank was prepared by performing the same treatment using a 5% phosphoric acid aqueous solution and distilled water instead of the 5% phosphoric acid aqueous solution containing 1% sulfanilamide and the 0.1% NED aqueous solution. A calibration curve was prepared using a sodium nitrite aqueous solution, and the concentration of nitrite ions in the culture cell supernatant after LPS stimulation was quantified to determine the NO production amount.

[0046] The results are shown in Figure 2. In Figure 2, each data represents the mean ± standard deviation, and different alphabets indicate significant differences (n = 4, P < 0.05, Tukey’s HSD test). Also, each symbol represents the following meanings. O: ethyl oleate, L: ethyl linoleate, Ln: ethyl α-linolenate, zO: ozonized ethyl oleate, zL: ozonized ethyl linoleate, zLn: ozonized ethyl α-linolenate.

[0047] As is clear from Fig. 2, among the non-ozonized fatty acid ethyl esters, only ethyl α-linolenate significantly suppressed NO production compared to the control. On the other hand, for the ozonized fatty acid ethyl esters, all of ethyl ozonized oleate, ethyl ozonized linoleate, and ethyl ozonized α-linolenate significantly suppressed NO production compared to the control. In particular, ethyl ozonized linoleate and ethyl ozonized α-linolenate significantly suppressed NO production to the same extent as in the case where the inflammatory reaction was not induced by LPS (LPS(-)). From these results, it was shown that the ozonized fatty acid ethyl esters have a superior NO production-suppressing effect compared to the non-ozonized fatty acid ethyl esters. Also, as the fatty acids of the ozonized fatty acid ethyl esters, linoleic acid, which is a divalent unsaturated fatty acid, and linolenic acid, which is a trivalent unsaturated fatty acid, were shown to have a superior NO production-suppressing effect compared to oleic acid, which is a monovalent unsaturated fatty acid. That is, it was suggested that ozonized polyvalent unsaturated fatty acid ethyl esters have a more excellent anti-inflammatory effect.

[0048] 2-3. Evaluation of mRNA Expression Levels of Inflammatory Factor Genes RAW264 cells were diluted in DMEM to a concentration of 5.0×10 5 cells / mL, and 500 μL of the diluted cells were seeded in a 24-well plate and incubated at 5% CO 2 , 37 °C for 24 hours. A mixture of 1 mL of DMEM medium containing 1% FBS added to 3 μL each of ethanol (non-treatment) and an ethanol solution of ozonized ethyl α-linolenate (about 1% ozonized ethyl α-linolenate) was used as the addition sample. After removing the medium in each well with an aspirator, 500 μL of the addition sample was added to each well, and the cells were incubated at 5% CO 2, incubated at 37 °C for 3 hours. The LPS stock (manufactured by Santa Cruz Biotechnology) was diluted 20-fold with DMEM containing 1% FBS to obtain a 500 ng / mL LPS solution. After adding 125 μL each of the 500 ng / mL LPS solution (LPS) or DMEM containing 1% FBS (non-treatment) to the wells, 5% CO 2 , incubated at 37 °C for 6 hours.

[0049] (RNA Extraction) After removing the medium with an aspirator, each well was washed with 500 μL of phosphate-buffered saline. 500 μL of Sepasol-RNA I Super G (manufactured by Nacalai Tesque) was added to each well and the collected solution was transferred to a 1.5 mL Eppendorf tube. To 500 μL of the collected material, 100 μL of chloroform was added and mixed by inverting for 15 seconds, followed by incubation at room temperature for 3 minutes and centrifugation at 10,800 rpm at 4 °C for 15 minutes. 120 μL of the upper layer was transferred to a new Eppendorf tube, 250 μL of 2-propanol was added and mixed by inverting, followed by centrifugation at 10,800 rpm at 4 °C for 10 minutes, and the supernatant was discarded by decanting. Further, 500 μL of 75% ethanol (prepared with DEPC-treated water) was added, centrifuged at 850 rpm at 4 °C for 15 minutes, and the supernatant was discarded. 10 μL of Nuclease-free Water was added and spun down, and incubated at 55 °C for 10 minutes. The RNA concentration was measured using NanoDrop Lite (manufactured by Thermo Fisher Scientific), and the diluted solution with Nuclease-free Water to a concentration of 0.5 ng / mL was used as the RNA solution.

[0050] (cDNA Synthesis) cDNA was prepared according to the protocol of ReverTra Ace qPCR RT Master Mix (manufactured by TOYOBO). 2 μL of a solution prepared by adding 8.8 μL of gDNA Remover to 440 μL of 4×DN Master Mix and 6 μL of RNA solution were mixed in a microtube and incubated at 37°C for 5 minutes. 2 μL of 5×RT Master Mix II was added, and the mixture was incubated at 37°C for 15 minutes, 50°C for 5 minutes, and 98°C for 5 minutes, and then rapidly cooled on ice to obtain a cDNA solution.

[0051] (Quantification of mRNA) Using cDNA and iQ SYBR Green Supermix (manufactured by Bio-Rad Laboratories), real-time quantitative PCR was performed using a thermal cycler. As inflammatory factor genes, the mRNA expression levels of TNFα, PTGS2, IL1β, and IL6 were evaluated. All data were normalized by the mRNA expression level of GADPH (Glyceraldehyde-3-Phosphate Dehydrogenase) as an internal standard. The primers used for quantification of each gene are shown in Table 3.

[0052]

Table 3

[0053] The results are shown in Figure 3. In Figure 3, each data represents the mean ± standard deviation, and is expressed as the relative expression level with the mean value of the mRNA expression level without ozonated ethyl α-linolenate treatment and with LPS treatment (「LPS」 in Figure 3) set to 100. 「non-treatment」 represents no ozonated ethyl α-linolenate treatment and no LPS treatment, and 「LPS+zLn」 represents ozonated ethyl α-linolenate treatment and LPS treatment. In the data of 「TNFα」, different alphabets indicate significant differences (n = 4, P < 0.05, Tukey’s HSD test). In the data of 「PTGS2」, 「IL1β」, and 「IL6」, 「*」 in Figure 3 indicates significant differences compared with 「LPS」 (n = 4, P < 0.05, Tukey’s HSD test). Also, in 「PTGS2」, 「IL1β」, and 「IL6」, 「non-treatment」 was below the detection limit and thus not described.

[0054] Although LPS treatment increased the mRNA expression of inflammatory factors TNFα, PTGS2, IL1β, and IL6, as is clear from Figure 3, further treatment with ozonated ethyl α-linolenate significantly suppressed their mRNA expression. These results suggest that ozonated ethyl α-linolenate has excellent anti-inflammatory effects.

[0055] 2-4. Evaluation of mRNA Expression Levels of Oxidative Stress Response Genes RAW264 cells were diluted in DMEM to a concentration of 5.0×10 5 cells / mL, seeded at 500 μL per well in a 24-well plate, and incubated at 5% CO 2 , 37 °C for 24 hours. Ethanol (non-treatment) and ethanol solutions of each ozonated fatty acid (ozonated oleic acid and ozonated α-linolenic acid) ethyl ester (each ozonated fatty acid ethyl ester approximately 1%) were prepared by adding 1 mL of 1% FBS-containing DMEM medium to 3 μL of each, and the resulting mixtures were used as the addition samples. After removing the medium from each well with an aspirator, 500 μL of each addition sample was added, and the cells were incubated at 5% CO2 It was incubated at 37°C for 3 hours.

[0056] Thereafter, RNA extraction, cDNA synthesis, and mRNA quantification were performed in the same manner as the method in the above “2-3.” The mRNA expression levels of HO-1 and NQO-1 were evaluated as oxidative stress response genes. All data were normalized by the mRNA expression level of GADPH as an internal standard. The primers used for quantification of each gene are shown in Table 4.

[0057]

Table 4

[0058] The results are shown in Figure 4. In Figure 4, each data represents the mean ± standard deviation, and is represented as a relative expression level with the mean value of the mRNA expression level without ozonated fatty acid ethyl ester treatment (“non-treatment” in the figure) set to 100. Different alphabets indicate significant differences (n = 4, P < 0.05, Tukey’s HSD test). Also, each symbol represents the following meaning. zO: Ozonated ethyl oleate, zLn: Ozonated ethyl α-linolenate.

[0059] As is clear from Figure 4, ozonated ethyl α-linolenate significantly increased the mRNA expression of HO-1. On the other hand, ozonated ethyl oleate did not significantly increase the mRNA expression of HO-1. Also, although not significant, ozonated ethyl α-linolenate tended to increase the mRNA expression of NQO-1 more than ozonated ethyl oleate. From these results, it was suggested that as the fatty acid of ozonated fatty acid ethyl ester, linolenic acid, which is a polyunsaturated fatty acid, has a superior antioxidant effect and thus a superior anti-inflammatory effect compared to oleic acid, which is a monounsaturated fatty acid.

[0060] 3. Examination of the effect of ozonated oils on enhancing HO-1 expression Triolein (TO), which is the main component of olive oil, and triglyceride (TG; oleic acid: linoleic acid = 19:1), which reflects the linoleic acid content in olive oil in the fatty acid composition, were ozonized, and their effects by oral ingestion were examined using mice.

[0061] 3-1. Preparation of Samples TO (≥99%) consisting only of oleic acid as the constituent fatty acid and TG (≥99%) with 95% of the constituent fatty acids being oleic acid and 5% being linoleic acid were both purchased from Tsukishima Food Industry Co., Ltd. 125 mL of dichloromethane (ultra-dehydrated) was added to TO or TG, and it was cooled to -20°C with acetone mixed with liquid nitrogen. Ozone gas generated from an ozone gas generator (ED-OG-R6, Eco Design Co., Ltd.) was bubbled into the reaction solvent at 1.0 L / min for 5 minutes. The ozone generation amount was set at 4 g / hr. Corn oil (manufactured by Fujifilm Wako Pure Chemical Corporation) was added to the obtained ozonized TO (zTO) or ozonized TG (zTG) in the ratio shown in Table 5, and the one with dichloromethane removed by an evaporator was used as a feed component.

[0062] 3-2. Feeding of Mice As test animals, db / db mice (5-week-old males, CLEA Japan, Inc.) were purchased and pre-fed for 1 week with the AIN-76 rodent standard feed composition (feed with 7% added corn oil) by the National Institute of Nutrition, USA. Then, 6-week-old mice were divided into the following groups (n = 6). · TO group: db / db mice were given 6.5% corn oil + 0.5% TO. · zTO group: db / db mice were given 6.5% corn oil + 0.5% zTG. · TG group: db / db mice were given 6.5% corn oil + 0.5% TG. · zTG group: db / db mice were given 6.5% corn oil + 0.5% zTG.

[0063] The test feed composition is as shown in Table 5. Each group of mice was individually fed each test feed using Rodent CAFE (manufactured by KBT Oriental Co., Ltd.) and bred for 4 weeks. Thereafter, growth parameters were measured (Table 6). On the last day of breeding, after fasting for 9 hours, the mice were sacrificed by cardiac blood collection under anesthesia, and the liver was excised for analysis.

[0064]

Table 5

[0065]

Table 6

[0066] 3-3. Evaluation of mRNA expression levels of oxidative stress response genes mRNA was extracted from 0.1 g of liver using the RNeasy Lipid tissue Mini kit (manufactured by QIAGEN). cDNA was prepared using Taqman Reverse Transcription reagents (manufactured by Thermo Fisher Scientific). Real-time RT-PCR was performed using the ABI Prism 7000 real-time PCR sequence detection system (manufactured by Thermo Fisher Scientific). TaqMan Universal PCR Master Mix (manufactured by Thermo Fisher Scientific) was used for gene amplification. Assay-on-Demand, Gene Expression Products (manufactured by Thermo Fisher Scientific) was used for gene quantification. The mRNA expression level of HO-1 (Taqman Assay ID: Mm00516005_m1) was evaluated as an oxidative stress response gene. The data was normalized by the mRNA expression level of 18s RNA (Taqman Assay ID: Mm04277571_s1) as an internal standard.

[0067] The results are shown in Fig. 5. In Fig. 5, each data represents the mean ± standard deviation, and is expressed as the relative expression level with the mean value of the mRNA expression level in the TO group set to 100. Different alphabets indicate significant differences (p < 0.05, Tukey’s HSD test). As is clear from Fig. 5, in the zTO group, the relative mRNA expression level of HO-1 did not increase significantly compared to the TO group. On the other hand, in the zTG group, the relative mRNA expression level of HO-1 increased significantly compared to any of the TO group, TG group, and zTO group. From these results, it was shown that triglyceride (oil and fat) containing ozonized polyunsaturated fatty acid as a constituent fatty acid exhibits an action of enhancing HO-1 expression in vivo.

Claims

1. An HO-1 expression enhancer comprising, as an active ingredient, an ozonized polyunsaturated fatty acid or an ester thereof, or an oil or fat having an ozonized polyunsaturated fatty acid as a constituent fatty acid.

2. The HO-1 expression enhancer according to claim 1, wherein the polyunsaturated fatty acid is linoleic acid or linolenic acid.

3. The HO-1 expression enhancer according to claim 1, wherein the oil is olive oil.

4. An anti-inflammatory agent which suppresses inflammation in the body by enhancing the expression of HO-1, comprising as an active ingredient an ozonized polyunsaturated fatty acid or an ester thereof, or an oil or fat having an ozonized polyunsaturated fatty acid as a constituent fatty acid.

5. An anti-inflammatory agent for inflammation in the body, comprising as an active ingredient an ozonized polyunsaturated fatty acid or its ester, or an oil or fat having an ozonized polyunsaturated fatty acid as a constituent fatty acid.

6. 6. The anti-inflammatory agent according to claim 4 or 5, wherein the polyunsaturated fatty acid is linoleic acid or linolenic acid.

7. 6. The anti-inflammatory agent according to claim 4 or 5, wherein the oil is olive oil.

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