JP2020103100A - Intracellular antioxidant function enhancing agent - Google Patents

Abstract

To provide a novel component that enhances an intracellular antioxidant function.SOLUTION: An intracellular antioxidant function enhancing agent containing hinokitiol, promotes expression of least one selected from the group consisting of thioredoxin reductase, thioredoxin, and sulfiredoxin to activate thioredoxin system, enhancing a cellular antioxidant function.SELECTED DRAWING: None

Description

The present invention relates to a novel component that enhances intracellular antioxidant function.

When cells are damaged by reactive oxygen species, a harmful reaction involving oxygen occurs, and when the DNA is damaged by this reaction, normal cell division is inhibited and aging and disease are caused. On the other hand, cells are also equipped with an antioxidant system that counteracts excessively generated reactive oxygen species, and the normal function of this antioxidant system repairs damage caused by reactive oxygen species. Typical examples of the antioxidant system include systems in which antioxidant enzymes such as Superoxide Dismutase (SOD) and Gulutathione Peroxidase (GPx) are involved.

Antioxidants are substances that reduce or eliminate harmful reactions involving reactive oxygen species, and vitamin E, vitamin C, vitamin B2, catechin, polyphenol, CoQ10, etc. are known. For example, vitamin B2 is known to act as a GPx coenzyme, and apple polyphenols have been reported to have SOD-like activity (Non-Patent Document 1).

On the other hand, when a pathogen infects a host, it causes an unwanted reaction (infectious disease) in the host. Antibacterial agents are substances that kill or suppress the growth of pathogens, and penicillin-based, cephem-based, carbapenem-based, monobactam-based, aminoglycoside-based, tetracycline-based, macrolide-based, lincomycin-based, glycopeptide-based antibacterial agents are known. ing.

It is known that hinokitiol, which is an unsaturated seven-membered monocyclic monoterpene contained in Hiba and the like, is also useful for treating infectious diseases caused by bacteria or molds in patients (Patent Document 1). It is used as a bactericide having a broad antibacterial spectrum. It has also been reported that hinokitiol is useful against vancomycin-resistant enterococci (Patent Document 2).

Proanthocyanidins: Food Sources, Antioxidant Properties and Health Benefits. Nova Science, p.p. 139-159 (2015)

International Publication No. 93/17559 JP 2001-131061 A

Antioxidants are used as various supplements in combination with health consciousness in recent years, and their usefulness as pharmaceuticals has also been proposed. If new antioxidants are discovered, further expansion of their availability can be expected.

Therefore, an object of the present invention is to provide a novel component that enhances the intracellular antioxidant function.

As a result of intensive studies, the present inventors have found that hinokitiol, which has not been known as an antioxidant until now, has an intracellular antioxidant function-enhancing action due to activation of the thioredoxin system. The present invention has been completed by further studies based on such findings.

That is, the present invention provides the inventions of the following modes.
Item 1. An intracellular antioxidant enhancer containing hinokitiol.
Item 2. Item 2. The antioxidant enhancer according to Item 1, which is a thioredoxin system activator.
Item 3. Item 3. The antioxidant function enhancer according to Item 1 or 2, which is at least one selected from the group consisting of a thioredoxin reductase expression promoter, a thioredoxin expression promoter and a sulfiredoxin expression promoter.

According to the present invention, a novel component that enhances the intracellular antioxidant function is provided.

The results (Comparative Example 1, Example 1, Reference Example 3, and Reference Example 4) obtained in the cell viability test of Test Example 1 are shown. The results (Reference Example 1 and Reference Example 2) obtained in the cell viability test of Test Example 1 are shown.

The intracellular antioxidant function-enhancing agent of the present invention is characterized by containing hinokitiol. Hereinafter, the intracellular antioxidant function enhancer of the present invention will be described in detail.

Hinokitiol Hinokitiol is an unsaturated seven-membered monocyclic monoterpene derived from natural products. Hinokitiol is a known component known to have a bactericidal action with low toxicity and a broad antibacterial spectrum. In the intracellular antioxidant function enhancer of the present invention, hinokitiol is used for a new purpose of enhancing the intracellular antioxidant function. In the present invention, hinokitiol may be a natural product obtained from a plant or a chemically synthesized product.

Examples of the natural raw material plant of hinokitiol include asunaro (Thujopsis dolabrata) and Taiwan cypress (Chamaecyparis taiwanensis). The plant may be a plant produced by cultivation or a plant collected from nature. The intracellular antioxidant function enhancer of the present invention may be contained in the form of an isolated and purified product of hinokitiol from a natural product, or may be contained in the form of a crudely purified product of hinokitiol from a natural product. .. Examples of the crude product include processed products of natural products, preferably hinokitiol-containing essential oil such as hiba oil, solvent extract of hinokitiol-containing essential oil, hinokitiol fraction of solvent extract and the like. Hiba oil is preferred as the essential oil. Extraction and isolation and purification of hinokitiol from the raw material plant can be carried out by known methods.

Also, a chemically synthesized product of hinokitiol can be obtained by a known method. Specifically, a method of synthesizing from dicyclopentadiene is known.

The blending amount of hinokitiol in the antioxidant of the present invention is appropriately set depending on the formulation form and application of the intracellular antioxidant, but is usually 0.0001 to 0.5% by volume. Are listed. From the viewpoint of further increasing the intracellular antioxidant function-enhancing effect, the amount of hinokitiol contained in the antioxidant-function promoter is preferably 0.0003 to 0.5% by weight, more preferably 0.0003 to 0.3% by weight. %, and more preferably 0.0003 to 0.2% by weight.

Other Ingredients The intracellular antioxidant function enhancer of the present invention may contain, in addition to hinokitiol, other ingredients depending on the application form, as long as the effects of the present invention are not impaired. Examples of such components include antiseptics, bactericides, antibacterial agents, antiphlogistics, antihistamines, local anesthetics, blood circulation promoters, thickeners, wetting agents, excipients, fragrances, sweeteners, and cooling agents. , Dyes, deodorants, surfactants, solvents, pH adjusters and the like.

Formulation Form The intracellular antioxidant function enhancer of the present invention is not particularly limited in its form and properties as long as it contains the above-mentioned hinokitiol. The form of the intracellular antioxidant function enhancer of the present invention may be either an external preparation or an internal preparation, and may be a drug (including quasi drug), cosmetic, food (functional food). , Foods for health and health, foods for patients, etc.), and the like. Furthermore, the properties of the intracellular antioxidant function-enhancing agent of the present invention are not particularly limited, and may be liquid, solid, semisolid (gel, ointment, paste) or the like.

Specific examples of the dosage form of the intracellular antioxidant function enhancer of the present invention include topical lotions, emulsions, ointments, creams, nasal drops, transdermal absorption agents, suppositories, oral hygiene agents, etc. Agents: injections, infusions such as drops, liquids, beverages, emulsions, suspensions, spirits, syrups, elixirs, tablets, pills, powders, fine granules, granules, tablets, capsules Oral medications such as

Production method The method for producing an intracellular antioxidant function enhancer of the present invention, using the above-mentioned hinokitiol and other components optionally blended, according to various forms and properties, and the purpose of use, conventionally known The usual formulation procedure in 1. may be followed.

Use The intracellular antioxidant function enhancer of the present invention, by administration, approaches the antioxidant system possessed by the cells to activate the antioxidant system itself. As a result, the cell defense function against oxidative stress due to reactive oxygen species can be improved.

The intracellular antioxidant function enhancer of the present invention can activate the thioredoxin system possessed by cells. That is, the intracellular antioxidant function enhancer of the present invention can be used as a thioredoxin system activator.

The thioredoxin system is one of intracellular antioxidant mechanisms constructed by thioredoxin reductase, thioredoxin, sulfiredoxin, peroxiredoxin and the like. Thioredoxin reductase converts thioredoxin into a reduced form (active form), thioredoxin converts oxidized proteins and peroxiredoxin into a reduced form, and sulfiredoxin converts oxidized peroxiredoxin into a reduced form. Reduced peroxiredoxin converts hydrogen peroxide, which is a reactive oxygen species, into water.

The intracellular antioxidant function enhancer of the present invention can activate the thioredoxin system by promoting the expression of thioredoxin reductase, thioredoxin, and sulfiredoxin involved in the thioredoxin system. That is, the intracellular antioxidant function enhancer of the present invention can be used as at least one selected from the group consisting of thioredoxin expression promoters, thioredoxin reductase expression promoters, and sulfiredoxin expression promoters. ..

The oxidative stress to which cells to which the intracellular anti-oxidant function enhancer of the present invention is applied is exposed to oxidative stress that occurs in every scene of daily activities, and oxidative stress that occurs temporarily and oxidative stress that occurs chronically. Both are included. The place of application of the intracellular anti-oxidant function enhancer of the present invention is not particularly limited as long as it is a place where cells exposed to oxidative stress are present, and examples thereof include skin, mucous membranes, organs and the like.

Specifically, the intracellular antioxidant function-enhancing agent of the present invention can be used for the prevention or treatment of pathological conditions caused by oxidative stress. More specifically, the intracellular antioxidant function enhancer of the present invention can be used for controlling oxidative stress in the prevention or treatment of lifestyle-related diseases and the prevention of aging.

The dose of the intracellular antioxidant function-enhancing agent of the present invention may be appropriately set according to the formulation form and the degree of symptoms to be applied. For example, in the case of an external preparation, it can be applied to the skin or mucous membrane several times a day. Specifically, it is possible to apply hinokitiol in an amount of 0.002 to 2 mg per one time, at a frequency of about once to several times a day.

Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited thereto.

Test Example 1: Cell viability test As a test cell, gingival fibroblast HGF (manufactured by ScienCell Research Laboratories) was used, and 10% by weight of fetal bovine serum manufactured by Corning was added to DMEM/HamF12 medium manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd. A collagen-coated 96-well plate with a medium was seeded with 10,000 cells/well and a medium amount of 100 μL/well. After culturing at 37° C. and 5% CO ₂ to a sub-confluent state, the medium was exchanged with the culture medium shown in Table 1 and cultivated for 6 hours. Then, the medium was exchanged with DMEM/HamF12 medium containing 10% fetal bovine serum, and culturing was continued for 48 hours. It was After completion of the culture, the medium was replaced with a medium containing 10% of fetal bovine serum in DMEM/HamF12 plus 10% of Alamar Blue. Two hours later, the absorbance at 600 nm (reference) and the absorbance at 570 nm were measured with a plate reader, and the cell viability was calculated by setting the condition of the medium of Reference Example 1 in Table 1 to 100%. As hinokitiol, hinokitiol manufactured by Takasago International Corporation is used, and 20 μM hinokitiol in the medium corresponds to 0.0003% by weight hinokitiol in the medium. As auranofin, auranofin manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd. was used. Auranofin reduces or stops the function of the thioredoxin system by inhibiting thioredoxin reductase. Thioredoxin reductase is a component that constructs the thioredoxin system, which is one of intracellular antioxidant systems.

The results are shown in FIGS. 1 and 2. In the figure, the vertical axis represents cell survival rate, and statistical analysis was performed by Tukey's test. As shown in Comparative Example 1, exposure of the cells to hydrogen peroxide almost killed the cells. On the other hand, as shown in Example 1, coexistence of hinokitiol with hydrogen peroxide in a cell-exposed environment resulted in increased cell viability. Significantly improved. In addition, as shown in Reference Examples 3 and 4, when a thioredoxin reductase inhibitor coexists with hydrogen peroxide and hinokitiol in a cell-exposed environment, the viability of cells is remarkably reduced again, and the extent of the reduction is It depended on the concentration of the inhibitor. This means that the thioredoxin reductase is inhibited and thus the antioxidant effect of hinokitiol does not work. As shown in Reference Example 2, the cell viability was similar to that of the control (Reference Example 1) even when auranofin was allowed to coexist in a cell-exposed environment in which hydrogen peroxide did not exist. That is, auranofin itself did not affect cell viability in the absence of hydrogen peroxide. From these results, it was shown that hinokitiol improved the cell viability by activating the thioredoxin system.

Test Example 2: RNA-Seq gene expression level test Using gingival fibroblast HGF (manufactured by ScienCell Research Laboratories) as a test cell, 10 parts by weight of Corning fetal bovine serum manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. in DMEM/HamF12 medium was used. % Of the added medium was seeded in a 5 cm Petri dish with 2 million cells and 3.5 mL of the medium. After culturing at 37° C. and 5% CO 2 to a sub-confluent state, the medium was replaced with the one shown in Table 2 and the cells were cultured for 24 hours. After completion of the culturing, total RNA was collected from the cells using Total RNA Extraction Miniprep System manufactured by Viogene, and a library was prepared using TruSeq Stranded mRNA Sample Prep Kit manufactured by illumina. 50-base single read sequencing was performed on the prepared library using a HiSeq 2500 sequencer manufactured by illlumina. The obtained reads were mapped to the human genome (GRCh38) using STAR, the number of mapped reads was counted for each gene, and statistical analysis of expression comparison between the two groups was performed using DESeq2. Specifically, the gene expression levels of Example 2 were compared with Comparative Example 2 as a reference, and the results obtained by analyzing the fold change (Fold Change) of the marker gene (Symbol) of the antioxidant system shown in Table 3 are shown in Table 3. Indicated.

As shown in Table 3, thioredoxin reductase TXNRD1 that converts thioredoxin into a reduced form (active form), thioredoxin TXN that converts oxidized protein or peroxiredoxin into a reduced form, and convert oxidized peroxiredoxin into a reduced form It was confirmed that the expression of sulfidoredoxin SRXN1 was promoted 1.9 times, 1.2 times and 2.5 times, respectively. On the other hand, with respect to the enzymes constructing other antioxidant systems such as SOD, GPX and CAT, the expression change was 0.8 to 1.1 times, and no obvious promoting action was observed. That is, it was shown that hinokitiol promotes the expression of thioredoxin reductase, thioredoxin, and/or sulfiredoxin.

Claims (3)

An intracellular antioxidant enhancer containing hinokitiol. The antioxidant function enhancer according to claim 1, which is a thioredoxin system activator. The antioxidant function enhancer according to claim 1 or 2, which is at least one selected from the group consisting of a thioredoxin reductase expression promoter, a thioredoxin expression promoter, and a sulfiredoxin expression promoter.