JP6333026B2 - Nitric oxide scavenger - Google Patents

Description

The present invention relates to a highly safe nitric oxide (hereinafter, sometimes referred to as “NO”) scavenger containing an extract of Aquinowaceae belonging to the genus Forget-me-not. More specifically, as a prophylactic / therapeutic agent for diseases such as skin diseases caused by increased NO production, coronary arteriosclerosis, angina pectoris, heart failure, hypertension, arteriosclerosis, septic shock, cirrhosis, renal failure, inflammatory diseases, etc. The present invention relates to useful pharmaceuticals, foods, and cosmetic compositions.

NO which is one of the active oxygens is a kind of nitrogen oxide (NOx) which is a problem as an air pollutant, and is oxidized into NO2 in the atmosphere. NO2 is said to be one of the causative substances of environmental pollution in addition to affecting respiratory organs at high concentrations. In addition, it has been pointed out that NO in the air may act as an oxidant, attacking unsaturated fatty acids contained in sebum, and causing rough skin (Non-Patent Document 1).

The cause of NO generation is mainly due to the combustion of fossil fuels such as oil, and is emitted from factories, automobiles, homes and the like. Moreover, it is known that it exists also in the smoke of tobacco as a more familiar example (nonpatent literature 2).

On the other hand, it is also known that NO exists in the living body. In vivo, NO is produced by NO synthase (hereinafter referred to as NOS) using L-arginine, NADPH, tetrahydrobiopterin, calmodulin and the like as a substrate, and functions as an extremely important physiologically active molecule. For example, the true identity of vascular endothelium-derived relaxing factor (EDRF) is NO, and it is also known that it plays a role of a foreign body attack in the information system in the nervous system and in the immune system.

Although NO has been considered to always act in a cytoprotective manner mainly from the viewpoint of blood flow retention action from the beginning of its discovery, high concentrations of NO may have cytotoxicity or adversely affect human pathology. It has been reported. The existence of endotoxin shock and arteriosclerosis has been clarified as an overproduction state of NO. In addition, the involvement of NO in many pathological conditions such as heart failure, myocarditis, septic shock, and cirrhosis has been reported (Non-patent Document 3).

L-arginine derivatives such as Nω-monomethyl-L-arginine, Nω-dimethyl-L-arginine, Nω-nitro-L-arginine, aminoguanidine and S-ethyl NOS inhibitors that inactivate NOS, such as arginine analogs such as isothiourea, NO scavengers that react rapidly with NO, such as iron complexes of diethylenetriaminepentaacetic acid, iron complexes of desferrioxamine, hydroxocobalamin, etc. (Non-Patent Document 4).

However, in clinical trials in which a NOS inhibitor as described above is administered, although it leads to improvement of symptoms, it is difficult to quantitatively control NO produced by controlling NO synthesis, resulting in increased blood pressure, organ blood flow. Problems have been pointed out that cause various side effects such as reduction and disability. Further, the conventional NO erasing agent as described above has not been focused on selectively eliminating NO.

Previously active oxygen range of reactive species generated in the energy metabolism and infection protective process in vivo ^{_{(O 2 -, H 2 O}} 2 , etc.), and treated as hazardous substances that cause oxygen toxicity far (Active oxygen toxicity theory).

On the other hand, there is an enzyme group called Nox (and Duox) in the living body that actively produces active oxygen not only in the antibacterial action of leukocytes but also in various cells such as vascular, epithelial / endocrine cells and lymphocytes. In recent years, it has been reported that physiological functions other than bactericidal action are exerted, and active oxygen also controls important information transmission as a cell protection signal in vivo (Non-patent Document 5).

Thus, it has been found that active oxygen also has a very important signal function in cells and tissues. Therefore, when trying to improve the overproduction state of NO, which has a risk of adversely affecting the body, using conventional antioxidants, the active oxygen that plays an important role for the living body such as cytoprotective signals is also eliminated. Will end up. For this reason, it is very important to selectively erase NO without erasing other active oxygen, and research focusing on NO is very meaningful.

Yoichi Okubo et al .: Journal of Air Pollution Society, 25 (3), 212-220, 1990 Shinagawa, K .; et al: Biochem. Biophys. Res. Commun, 253, 99-103, (1998) Nihon Kagaku: Quarterly Chemical Review NO-Chemistry and Biology, p. 161-168 Tetsuhiko Yoshimura: “NO (Nitric Oxide) From Space to Cells”, p113-134, Kyoritsu Shuppan (1998) Hideki Sumimoto, FEBS Journal, Volume 275, Issue 13 (2008)

An object of the present invention is to provide a substance having high activity for selectively eliminating NO.

In the present invention, among active oxygen species, a component that selectively eliminates nitric oxide is an effective component.
Specifically, the above-mentioned problem is solved by using an extract of Achillea regusa as an active ingredient and using a nitric oxide scavenger. The present nitric oxide scavenger can be used as it is or in addition to foods, drinks, pharmaceuticals, or cosmetics.

The nitric oxide scavenger of the present invention is for skin diseases caused by increased NO production and disease symptoms such as coronary arteriosclerosis, angina pectoris, heart failure, hypertension, arteriosclerosis, septic shock, cirrhosis, renal failure, and inflammatory diseases. It is also useful as a pharmaceutical, food or cosmetic composition useful as a preventive or therapeutic agent.

Results of radical scavenging ability test Results of NO erasing ability evaluation test

The Achillea forget-me-not used in the present invention is a perennial plant belonging to the genus Forget-me-not. The site to be used is not particularly limited. In addition to whole plants, flowers, leaves, stems, roots, pods, seeds, etc. can be used.

Although the preparation method of an Aquinoa legusa extract is not specifically limited, For example, it extracts under low temperature from heating using various suitable organic solvents. Examples of the extraction solvent include water; lower monohydric alcohols such as methyl alcohol and ethyl alcohol; liquid polyhydric alcohols such as glycerin, propylene glycol, and 1,3-butylene glycol; ketones such as acetone and methyl ethyl ketone; and ethyl acetate. One type or two or more types of alkyl esters; hydrocarbons such as benzene and hexane; ethers such as diethyl ether; halogenated alkanes such as dichloromethane and chloroform can be used. Among them, water, ethyl alcohol, and a mixed solvent of one or more of 1,3-butylene glycol are particularly suitable.

Although the extraction method of the plant extract which can be used for this invention is not specifically limited, For example, if it is dried, it is 1-1000 times amount by mass ratio, especially 10-100 times amount of solvent, 0 degreeC or more, In particular, it is preferably performed at 20 ° C. to 40 ° C. for 1 hour or longer, particularly 3 to 7 days. Moreover, you may heat-extract at 60-100 degreeC for 1 hour.

Each of the above-mentioned extracts obtained under the above conditions may be used as an extracted solution, but if necessary, use a product that has been subjected to a treatment such as filtration, concentrated and powdered as appropriate. Can do.

The effective amount of the plant extract in the nitric oxide scavenger of the present invention may be present.
Generally, it is preferably 0.0001 to 100% by mass in terms of the evaporated and dried content with respect to the composition of the nitric oxide scavenger, and particularly the range of 0.01 to 10.0% by mass is optimal.

The dosage form of the nitric oxide scavenger of the present invention is not particularly limited. Various dosage forms such as liquid, gel, powder, and emulsion can be used.

The nitric oxide scavenger of the present invention can be produced by blending the nitric oxide scavenger of the present invention into a base. In addition to the nitric oxide scavenger of the present invention, as long as the effects of the present invention are not impaired, for example, fats and oils, waxes, hydrocarbon oils, ester oils, higher alcohols, silicone oils, ultraviolet absorbers, ultraviolet scattering agents, moisturizing agents. Agent, surfactant, water-soluble polymer, thickener, powder, various active ingredients, skin protectant, anti-aging agent, plant extract, antiseptic, anti-inflammatory agent, pH adjuster, sequestering agent, oxidation An inhibitor or the like can be blended.

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples. Unless otherwise specified, the blending amount is expressed in mass%.

<Preparation of test substance>
(1) The plant extract of the test substance was prepared by using a dried product of Akinowasusa (a mixture of leaves and stems), summer hay (a mixture of leaves and stems), and radish (leaves). 10 g of 50% ethanol of 10 times the amount was added to 1 g of each plant raw material and extracted by heating at 60 ° C. for 3 hours. After extraction, the mixture was filtered to remove the plant material, and diluted with distilled water so that the solid evaporation residue was 1%. By the above operation, a plant extract prepared so that the solid evaporation residue was 1% was obtained.
(2) Ascorbic acid, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) and BHT (dibutylhydroxytoluene) were dissolved in DMSO (dimethyl sulfoxide), and all were 10 mM. Concentration was adjusted.

DPPH radical scavenging test In order to confirm the radical scavenging ability of known antioxidants and plant extracts, the DPPH radical scavenging test was used to evaluate the radical scavenging ability.
(1) 8.53 g of MES (2-morpholinoethanesulfonic acid) was dissolved in 100 mL of dH2O to prepare 400 mM MES.
(2) 0.0628 g of DPPH (1,1-diphenyl-2-picrylhydrazyl) was dissolved in 100 mL of EtOH to prepare 0.4 mM DPPH.
(3) DPPH: MES: dH2O = 4: 1: 3 was mixed, and this was prepared as DPPH Working Solution.
(4) 10 μL of the plant extract prepared by the above method was dispensed to a 96-well microplate, 190 μL of DPPH Working solution was added, and the plate was shaken for 30 minutes with a plate mixer.
(5) Thereafter, the absorbance at 540 nm was measured with a microplate reader. After the measurement, it was calculated using the following formula for calculating the radical scavenging rate. The results are shown in FIG. In addition, only the plant name is described in the plant extract.

Formula for calculating radical scavenging rate (1) 10 mM Trolox was prepared, serially diluted with DMSO, and used as a standard reagent.
(2) A calibration curve (Y = aX + b, Y = absorbance, X = concentration) was prepared from the values and concentrations of the Trolox absorbance Ab540 of the above serial dilution.
(3) From the calibration curve Y = aX + b, the concentration was calculated as the Trolox equivalent of the sample.
<Formula 1>
Trolox equivalent = (measurement sample Ab540-b) / a (mM)

Ascorbic acid and BHT, which are known antioxidants, showed high radical scavenging ability, and Trolox equivalents were 8.5 mM and 5.7 mM, respectively, at 10 mM. The radish extract and summer hay extract were also confirmed to have the same high radical scavenging ability as that of ascorbic acid and BHT, while the extract of Achillea grass extract was very low in radical scavenging compared to the above-mentioned known antioxidants. Only the ability was confirmed.

NO scavenging ability evaluation test In order to confirm the NO scavenging ability of known antioxidants and plant extracts, NO scavenging ability is measured using the Griess method, which measures the amount of NO by measuring NO2-an oxide of NO. Was evaluated. For the measurement by the Griess method, NO2 / NO3 Assay Kit-CII (Colorimetric) (manufactured by Dojin Chemical) was used. As NO generator, NOC-7 (1-hydroxy-2-oxo-3- (N-methyl-3-aminopropyl) -3-methyl-1-triazene) (manufactured by Dojin Chemical Co., Ltd.) was used immediately before use. Prepared with an alkaline solution of 0.1N sodium hydroxide. Developed as a nitric oxide donor, NOC-7 is an unstable compound that is stable in alkaline solution but produces nitric oxide under neutral and acidic conditions (half-life of 5 at 25 ° C, pH 7.4). Min).

(1) To a 96-well microplate was added 10 μL of PBS, 40 μL of the buffer solution (pH = 7.6), and 10 μL of the measurement sample prepared in [0025].
(2) Thereafter, 40 μL of a NOC-7 solution prepared to 100 μM was added and reacted at room temperature for 30 minutes.
(3) After completion of the reaction, 50 μL of Griess reagent A in Kit was added, followed by 50 μL of Griess reagent B and left for 10 minutes to perform a color reaction.
(4) Absorbance at 540 nm was measured with a microplate reader. The absorbance in each measurement sample is S540, the absorbance of a control in which only buffer solution and NOC-7 are mixed instead of the sample is C540, and the absorbance of a blank not containing NOC-7 is B540. And the results are shown in FIG.

<Formula 2>
NO erasure rate (%) = {100 × (1− (S540−B540) / (C540−B540)}

Trolox, which is known as an antioxidant, was observed to have a high NO scavenging ability with a NO suppression rate of 40%, but NO scavenging ability was not observed in two types of BHT and ascorbic acid that obtained high radical scavenging ability. On the other hand, in the Aquinois regusa extract in which the radical scavenging ability was not observed, a high NO suppression rate of 47% was obtained.
In the examples, only 50% ethanol extract of leaves and stems of Achillea grass is disclosed, but the same tendency was observed in other parts and extraction solvents.

From the above results, it has been clarified that the extract of Achillea regusa of the present invention selectively eliminates NO without removing other active oxygen species. This extract of Achillea regusa selectively eliminates excess NO present in the living body, and when it is predicted that excessive NO will be generated and the effect of obtaining normal NO concentration, it is excessively administered in the living body. It has the effect of preventing the generation of NO. Therefore, prophylactic / therapeutic agents for skin diseases such as rough skin caused by increased NO production and disease symptoms such as coronary arteriosclerosis, angina pectoris, heart failure, hypertension, arteriosclerosis, septic shock, cirrhosis, renal failure, inflammatory diseases, etc. Can be used for useful pharmaceuticals, foods, cosmetics and the like.

Claims (1)

A nitric oxide scavenger characterized by containing an extract of leaves and / or stems of Achillea forget-me-not belonging to the genus Forget-me-not.

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