JP2007320859A - Inhibitor on expression of enzyme producing active oxygen species - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an inhibitor on the expression of an enzyme producing active oxygen species. <P>SOLUTION: The inhibitor on the expression of the enzyme producing active oxygen species contains brassinin and its analogue. The enzyme producing the active oxygen species is NADPH oxidase or xanthine oxidase. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention provides an active oxygen-producing enzyme expression inhibitor.

  In daily life, reactive oxygen species such as superoxide anion radical, hydroxyl radical radical, hydrogen peroxide, singlet oxygen, etc. are produced in the body. Reactive oxygen is produced in cells along with oxidative energy metabolism by mitochondria and drug metabolism. On the other hand, the amount of active oxygen produced by leukocytes, especially neutrophils, is the largest in the living body, and it is known that active oxygen is used for immune responses such as sterilization and foreign substance removal. Active oxygen is highly reactive in nature and oxidizes others in a very short time and disappears itself. It is known that when it is overproduced, it has a harmful effect due to oxidation, and the living body avoids this risk by producing enzymes and bioactive substances that eliminate excess active oxygen.

  On the other hand, active oxygen tends to be excessively produced in the body due to stress, ultraviolet rays, smoking, food additives, drinking, intense exercise, and the like, and the ability to erase excess active oxygen tends to be insufficient due to individual differences and aging. Then, reactive oxygen rich in reactivity reacts with lipids contained in the cell membrane, for example, unsaturated fatty acids that are easily oxidized to form lipid peroxides. Cell membrane damage progresses due to the formation of lipid peroxide, and the proteins constituting the cell membrane are denatured by peroxidation, causing damage to the structure and function of tissues and organs. In particular, blood vessels are organs that have many opportunities to be affected from outside the body, and they are always subjected to physical and chemical stimuli by transporting substances essential for life activity, such as nutrients and metabolites, to each organ. The frequency of exposure to reactive oxygen species produced by leukocytes for the purpose of excluding foreign substances and reactive oxygen species produced by vascular endothelium in response to physiologically active substances transferred via blood vessels is high. As described above, the blood vessel wall is easily damaged, which increases the risk of thrombosis.

  For this reason, excessive active oxygen produced in the body has been reported to increase the risk of life-style related diseases such as malignant tumors, myocardial infarction, cerebral infarction, diabetes, and allergic symptoms, and accelerate their progression. (Non-patent document 1) In the field of foods and pharmaceuticals, research and development on active oxygen production inhibitors are being promoted for the prevention and treatment of symptoms and diseases induced or promoted by excessive production of active oxygen.

  Substances that suppress the production of active oxygen include enzyme inhibitors that inhibit free radical-producing enzymes (eg, NAD (P) H oxidase (NAD (P) H oxidase), xanthine oxidase), etc. Substances that suppress the expression of these enzymes are listed, and research is being conducted. For example, an isopropyl ester compound of 6-cyano-5-methoxycarbonyl-2-methyl-4- (3-nitrophenyl) -1,4-dihydropyridine-3-carboxylic acid is known as a NADPH oxidase expression inhibitor. (Patent Document 1). Moreover, quercetin and its glycoside (Patent Document 2) are known as xanthine oxidase inhibitors.

On the other hand, brassine and its analogs are indole produced by cruciferous plants such as Chinese cabbage, cabbage, rapeseed, mustard vegetable, broccoli, cauliflower, kale, kohlrabi, hata-daikon, turnip, rutabaga (kabuha button), etc. It is a derivative and is known as phytoalexin for combating stress such as invasion of microorganisms (Non-patent Document 2). Antibacterial action (Non-Patent Document 3) and anticancer action (Non-Patent Document 4) are known as actions of brassin, but it is not known at all to suppress the production of active oxygen.
JP 2005-232059 A JP 2003-171283 A Itakura Hiroshige, Nutrition-Evaluation and Treatment, Vol.19, N0.3, p293-298, 2002 Soledade C. Pedras, Sabine Montaut et al, J. Org. Chem. Vol. 69, No. 21, p4471-4476, 2004 Mitsuo Takasugi, Nobukatsu Katsui and Akira Shirata, J. Chem. Soc. Chem. Commun., 1986, p1077-1078 Rajendra G. Metha, et al., Carcinogenesis, Vol.16, No.2, 1995, Feb, p399-404

  An object of the present invention is to provide an active oxygen-producing enzyme expression inhibitor, an antioxidant, and the like that can suppress excessive active oxygen in the body.

  The inventors of the present invention have searched for active oxygen-producing enzyme expression-suppressing substances and found that brassinin and its analogs have an excellent activity-suppressing effect on active oxygen-producing enzyme.

  That is, the present invention provides an active oxygen-producing enzyme expression inhibitor containing brassin and its analogs.

  Since the present invention can suppress excessive production of active oxygen in the body, it can be used for prevention, amelioration, or treatment of symptoms or diseases induced or promoted by excessive production of active oxygen, including lifestyle-related diseases. it can.

  In the present invention, brassinin (brassin, 3-S-methyldihiocarbamoyl aminomethyl indole) and its analogs are: It is an indole derivative produced by cruciferous plants such as turnip and turnipaga. Specifically, brassinin (compound 1), 1-methoxybrassinin (1-methoxybrassinin, compound 2), 4-methoxybrassin (4-methoxybrassinin, compound 3), brassin (brassitin, compound 4), 4-methoxy Dihydrocyclobrassin (4-methoxydehydrocyclobrassinin, compound 5), Isalexin (isalexin, compound 6), Brush canate A (brassicanate A, compound 7), Rutalexin (compound 8), cyclobrassinone (compound 9), spiro Examples include brassine (spirobrassinin, compound 10), brass canal A (compound 11), and brass lexin (compound 12). More preferred is brassin.

Such a brassin and its analog can be obtained from a natural product or callus containing the brassin and its analog by a known extraction method (Non-patent Documents 2, 3 and 4). Alternatively, a glycoside bound to brassin and its analogs, a natural product containing a derivative or precursor of these glycosides, a callus or the like, and a known organic chemical synthesis method. Brassine and its analogs may be obtained.
Examples of natural products or callus containing brassinin and its analogs, or derivatives or precursors thereof, such as glycosides, include cruciferous plants, specifically Chinese cabbage, cabbage Chinese cabbage, cabbage, rapeseed, mustard vegetable. , Broccoli, cauliflower, kale, kohlrabi, hatsudaikon, turnip, turnipaga (kabuha button) and the like.
Brassin and its analogs can also be obtained by known organic chemical synthesis methods (Non-Patent Documents 2 and 4).

Further, the purity of brassinin and its analogs may be increased by appropriately combining known separation and purification methods with the obtained synthesized product or extract.
Brassine and its analogs obtained by the above synthesis and extraction can be roughly purified as long as they meet the standards acceptable on pharmaceuticals or foods by single or multi-step separation and purification, etc. and exhibit the effects of the present invention. It may be a thing.
Specifically, the extraction may be performed using water such as water, hot water, alcohol water and / or polar and nonpolar solvents such as organic solvents. Separation and purification may be performed using organic solvent precipitation, centrifugation, ultrafiltration membrane, high performance liquid chromatograph, column chromatograph, or the like.

The active oxygen producing enzyme in the present invention means an enzyme that produces active oxygen generated in a living body. For example, NAD (P) H oxidase (NAD (P) H oxidase), xanthine oxidase, liposomal Oxygenase (Lipooxygenase), cyclooxygenase (Cyclooxgenase), etc. are mentioned, More preferably, they are NAD (P) H oxidase and xanthine oxidase.
Antioxidation in the present invention refers to attenuating cell / tissue damage due to excessive production of active oxygen.

Here, the active oxygen is a radical such as a superoxide anion radical or a hydroxy radical, hydrogen peroxide, singlet oxygen, nitric oxide, nitrogen dioxide, ozone, lipid peroxide (LOOH or LOO · radical, etc.), halogen, and the like. Oxygenated oxygen and the like can be mentioned, preferably a radical, more preferably a superoxide anion radical, a hydroxyl radical or a lipid peroxide radical, and particularly preferably a superoxide anion radical.
In the present invention, the cause of excessive production of active oxygen includes stress, ultraviolet rays, smoking, food additives, drinking, intense exercise, and the like.

  As shown in Examples described later, brassin can suppress the expression of an active oxygen producing enzyme. Therefore, brassine and its analogs are active oxygen-producing enzyme expression inhibitors and antioxidants containing them as active ingredients, and preventive, ameliorating or treating agents or symptoms or diseases induced or promoted by excessive production of active oxygen (hereinafter referred to as “the active oxygen producing enzyme”). And an active oxygen-producing enzyme expression inhibitor, etc.), and can also be used to produce an active oxygen-producing enzyme expression inhibitor and the like. In addition, the active oxygen-producing enzyme expression inhibitor and the like can be used as beverages / foods, cosmetics, quasi-drugs, pharmaceuticals and the like for preventing, improving or treating the symptoms or diseases. Brassine and its analogs have a physiological function that promotes prevention, treatment, and improvement of lifestyle-related diseases, exhibiting an action to suppress the expression of active oxygen-producing enzymes in the body, and improving and preventing lifestyle-related diseases and aging. Therefore, it can be used as a special-purpose food or drink such as a food or drink or a cosmetic with a label indicating that it is used for the purpose.

Diseases induced or promoted by excessive production of active oxygen include, for example, cardiovascular diseases, cranial nervous system diseases, digestive system diseases, kidney diseases, respiratory diseases, metabolic / endocrine diseases, allergic diseases, ocular diseases, aging / Examples include senile diseases (Non-Patent Document 1).
Here, examples of the cardiovascular disease include arteriosclerosis, ischemic heart disease (arrhythmia, angina pectoris, myocardial infarction), hypertension, and cerebrovascular disease (cerebral infarction, cerebral hemorrhage). Examples of cranial nervous system diseases include Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, traumatic epilepsy, and senile dementia. Examples of digestive organ diseases include gastritis, gastric ulcer, enterocolitis, ulcerative colitis, irritable colitis, Crohn's disease, drug-induced colitis, cystitis, hepatitis, and cholecystitis. Examples of renal diseases include glomerulonephritis, diabetic nephritis, renal failure, and drug-induced nephropathy. Respiratory diseases include bronchial asthma, drug-induced pneumonitis, obstructive pulmonary disease. Metabolic / endocrine diseases include diabetes, hyperlipidemia, and menopause. Allergic diseases include chronic indirect rheumatism and systemic lupus erythematosus. Eye diseases include cataracts, diabetic retinopathy, retinopathy of prematurity, and age-related macular degeneration. Examples of aging / senile diseases include skin aging, spots and freckles. More preferred are cardiovascular diseases such as cardiovascular diseases and metabolic / endocrine diseases, particularly preferably arteriosclerosis, ischemic heart disease (arrhythmia, angina, myocardial infarction), hypertension, cerebrovascular disease (cerebral infarction, Cerebral hemorrhage), diabetes, hyperlipidemia, Alzheimer's disease.
Among these, circulatory system diseases, malignant tumors, digestive organ diseases, metabolic / endocrine diseases are said to be lifestyle-related diseases because they are deeply related to stress, smoking, eating and drinking, drinking alcohol, intense exercise, etc. These active oxygen-producing enzyme expression inhibitors are particularly effective for the prevention, amelioration, and treatment of the disease or symptoms associated therewith.

  When the active oxygen-producing enzyme expression inhibitor of the present invention is used as a beverage / food, for example, beverages such as fruit juice beverages, carbonated beverages, tea-based beverages, milk beverages, alcoholic beverages, soft drinks, jelly-like foods and various snacks , Baked confectionery, cakes, chocolate, gum, candy, soup, etc.

  In addition, when the active oxygen-producing enzyme expression inhibitor of the present invention is used as a pharmaceutical product, for example, it can be used as an oral liquid preparation such as a tablet, granule or the like, an oral solid molding agent, an internal solution or a syrup. Can do. Moreover, it can be used in various forms such as emulsified cosmetics, creams, milky lotions, lotions, gels, etc., in the form of medicinal skin external preparations such as ointments and cosmetic skin external preparations.

  Such active oxygen-producing enzyme expression inhibitors containing brassin and its analogs are mixed and dispersed together with a carrier that is directly or pharmaceutically acceptable by a general production method, and then processed into a desired form. Can be obtained by: In this case, in addition to the brassin and its analogs used in the present invention, oily bases such as vegetable oils and animal oils commonly used in such forms, analgesic anti-inflammatory agents, analgesics, bactericidal antiseptics, astringents, skin softening Agents, hormones, vitamins, moisturizers, UV absorbers, alcohols, chelating agents, pH adjusters, preservatives, thickeners, dyes, fragrances, etc. should be added as appropriate so long as they do not interfere with the effects of the present invention. Can do.

  The total blending amount of brassine and its analogs in the above-mentioned preparation of the present invention is usually 0.00001 to 20% by mass, particularly 0.00002 to 5% by mass, based on the total composition as a dried product.

Example 1: Inhibitory effect of brassinine on active oxygen production SD rats (10-12 weeks old, male) were used as animals. Blood was collected from the jugular vein under sevoflurane anesthesia, 1 mL of 10 μM brassin PBS solution or control solution (PBS) was added to 1 mL of blood, layered with a reagent for blood cell separation, centrifuged, and the leukocyte fraction was collected. did. To the collected leukocytes, 1 mL of 10 μM brassin PBS solution or control solution (PBS) is added again and reacted at room temperature for 1 hour, followed by addition of 10 μM of fluorescent reagent 5,6-CM-H ₂ DCFDA (Invitrogen) at room temperature. Reacted for 20 minutes. Further, 10% (v / v) of a fixing reagent (BECKMAN COULTER) was added and reacted at room temperature for 20 minutes, and then the amount of active oxygen in leukocytes was measured by flow cytometry (Becton Dickinson).

As shown in FIG. 1, the amount of active oxygen produced by leukocytes was significantly reduced by 35% in the brassin addition group compared to the control group.
Therefore, it is considered that brassin can suppress active oxygen produced in the body and has an antioxidant action.

Example 2: Inhibitory effect on expression of NAD (P) H oxidase and xanthine oxidase of brassin The expression levels of NAD (P) H oxidase and xanthine oxidase gene in leukocytes were measured by a quantitative PCR method using a TaqMan probe.
Expression mRNA was purified from the leukocytes of the brassin addition group and control group obtained in Example 1, and reverse transcription PCR was performed using this mRNA as a template to prepare cDNA. Quantitative PCR was performed using this cDNA.
Each expression level was measured using a primer specific to each of gp91phox and p47phox, which are subunits of NAD (P) H oxidase, and TaqMan probe (Applied Biosystems). The expression level was corrected by the expression level of GAPDH and expressed as a relative value when the control group was 100%.
Moreover, it replaced with the subunit of NAD (P) H oxidase also about the xanthine oxidase, and it performed by the same method.

As shown in FIG. 2, the brassin-added group had 44%, 53%, and 65% of the mRNA expression levels of gp91phox and p47phox of NAD (P) H oxidase and xanthine oxidase significantly compared to the control group, respectively. Declined.
Therefore, brassin can suppress production and expression of active oxygen (NADPH oxidase and xanthine oxidase). This means that production of superoxide anion radicals produced in the body can be suppressed, and production of a series of reactive oxygen species generated from this can be suppressed.
In addition, since the subunits gp91phox and p47phox are suppressed, it is considered that the production of NAD (P) H function using both as constituent molecules can also be suppressed.

Formulation Example (1) Coffee Drink Brassin and its Analogue 0.00002% by mass
5.5% by mass of coffee beans
Milk 7.0% by mass
Sugar 6.0% by mass
Perfume slightly baking soda (adjusted to pH 6.5)
Water balance

(2) Juice beverage brassine and its analog 0.0002 mass%
Vegetable juice 40% by mass
(Carrot, lemon, parsley, broccoli alone or in mixture)
Juice 40% by mass
(Apples, tangerines, Valencia oranges alone or as a mixture)
Acidulant appropriate amount Fragrance appropriate amount Vitamin C appropriate amount

(3) Candy brushin and its analog 0.002 mass%
Sucrose ester (emulsifier) 0.2% by mass
Minamata 35% by mass
35% by mass of sugar
Wheat flour 5% by mass
Condensed milk 17% by mass
Milk 6% by mass
2% by weight of butter
Perfume

The production amount of active oxygen in leukocytes of brassin when the control is 100% in SD rats is shown. The expression levels of NAD (P) H oxidase (gp91phox and p47phox) and xanthine oxidase as active oxygen producing enzymes when the control is 100% are shown.

Claims (5)

  An active oxygen-producing enzyme expression inhibitor containing brassin and its analogs.   The active oxygen-producing enzyme expression inhibitor according to claim 1, wherein the enzyme that produces active oxygen is NADPH oxidase or xanthine oxidase.   An antioxidant containing brassin and its analogs.   An agent for the prevention, amelioration, or treatment of a symptom or disease induced or promoted by overproduction of active oxygen containing brassin and its analogs.   A food / beverage product, which contains brassin and its analogs, has an action of suppressing the expression of active oxygen producing enzyme, and is labeled for use in preventing or improving lifestyle-related diseases.

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