JP2020132615A - TYROSINASE ACTIVITY INHIBITOR OF Nrf2 ACTIVATOR, AND WHITENING AND SKIN BEAUTY AGENT BY MELANIN PRODUCTION INHIBITORY ACTION - Google Patents

Abstract

To provide tyrosinase activity inhibitors containing as an active ingredient Nrf2 activator that is useful and highly safe, and to provide whitening and skin beauty agents having a melanin production inhibitory action.SOLUTION: Peucedanum japonicum (long-lived grass, Sakuna) extract, and pyranocoumarin compound (formula 1) contained thereof and the like are contained as active ingredients (where, Ris CHCO group, Ris CHCH=C(CH)CO group (cis isomer, trans isomer), (CH)C=CHCO group, (CH)CH-CHCO group, or CHCH-CH(CH) CO group, in a derivative, or Ris CHCH-CH(CH)CO group, and Ris CHCO group, in a compound).SELECTED DRAWING: None

Description

The present invention relates to a highly safe and highly effective Nrf2 activator in the fields of cosmetics, pharmaceuticals, health foods and functional foods, and a button Siler (long-lived grass, freckle) of the genus Umbelliferae, which contains the same. It relates to whitening and skin-beautifying effects by preventing liver spots such as spots and freckles due to melanin pigmentation caused by Nrf2 activator of the extract and pyranocoumarin compound and sun-induced pigmented spots, and prevention of melanoma (black species) disease.

While we aerobic organisms acquire high energy by utilizing oxygen, active oxygen (Reactive Oxygen Species, ROS) and active nitrogen (Reactive Nitrogen Species, RNS), which have strong oxidizing power, are generated in the living body. This cell damage caused by excessive ROS or RNS, or the peroxide generated, is an external factor of oxidative stress that causes various diseases such as arteriosclerosis, myocardial infarction, and carcinogenesis (Non-Patent Documents 1 and 2). .. Ultraviolet rays give oxidative stress with inflammation to the skin, and cause sun-induced pigmented spots and chloasma of freckles and melanoma of skin cancer due to excessive melanin production and pigmentation (Non-Patent Document 3).

In order to prevent various diseases caused by this oxidative stress, the antioxidant proteins NAD (P) H quinone oxidoreductase (NQO1), thioredoxin, hemuosigenase 1 (HO-1) and the detoxifying enzyme glutathione-S-transferase are used in vivo. Etc. are expressed to protect. In the expression of these enzymes and proteins, the transcription factor Nrf2, which binds to Keap1 in the cytoplasm, dissociates from Keap1 and translocates into the nucleus in response to oxidative stress and neoelectronic substances. Nrf-2 translocated into the nucleus binds to the antioxidant response sequence (ARE) and expresses the antioxidant protein HO-1 and detoxifying enzyme to protect cells and prolong survival (Non-Patent Document 4).

Kukoshi plant extracts containing an Nrf2 activator (Patent Document 1) and flavonoid hesperidin derivatives and sceptrin have been reported as Nrf2 activators (Patent Documents 2 and 3).

In the literature, the evaluation of Nrf2 activity has not yet demonstrated the expression level of antioxidant proteins associated with the activity. Further, in Document 3, it is extracted and separated from marine organisms, and there remains a quantitative problem in its practical use.

The inventor reported that the Nrf2 activator that solved the above problems was solved by the button bow extract and the pyranocoumarin compound contained therein (Non-Patent Document 5).

Ultraviolet rays cause oxidative stress with inflammation on the skin, which causes excessive melanin production and the resulting pigmentation of spots, sun-induced pigmented spots of freckles and chloasma, and melanoma of skin cancer. Melanin is produced by oxidative polymerization of tyrosine with tyrosinase. Pigmentation due to excessive melanin production causes stains and freckles and impairs beautiful skin. The accumulation also causes melanoma of skin cancer.

Conventionally, in order to reduce these stains and freckles, a whitening agent has been searched from nature using the inhibition of tyrosinase activity and the suppression of melanin production as an index (Patent Document 4), or an indole compound or the like has been synthesized (Patent Document 5). However, in reality, it is not suppressed by an in vivo control mechanism that suppresses inflammation accompanied by oxidative stress due to ultraviolet rays and accumulation of excess melanin. Therefore, it is due to a single enzyme inhibition, and a problem remains from the viewpoint of a sustained preventive effect.

The Nrf2 activator of the present invention is a whitening / skin-beautifying agent that is safe and has a long-lasting effect on the skin because it can control the suppression of inflammation associated with oxidative stress and the suppression of excess melanin from within the living body.

Button Siler, also known as long-lived grass or sakuna, is used as a traditional medicinal ingredient for the prevention of colds, asthma, and neuralgia. Recent studies have revealed that the contained pyranocoumarin compound (Formula 1) acts on adipocytes and has an anti-obesity effect that suppresses the synthesis of triglycerides (Non-Patent Document 6). In this way, the safety of using this material is guaranteed.

Published Japanese Patent Application Laid-Open No. 2012-2111107 Published Japanese Patent Application Laid-Open No. 2011-256126 Publication of Patent No. 2016-183111 Publication of Patent No. 2016-141657 Publication of Patent No. 2018-168142

Naoyuki Taniguchi, Takeshi Endo, Biochemistry of Oxidative Stress Redox, 2000 Hiroyuki Tsutsui, History of Medicine, Vol. 218, No. 1, 2006 Nordlund, et al. , The Pigmentary System: Physiology and Pathophysiology. , Ed. Oxford University Press, New York, 2006 Kaspar, et al. , Free Radical Biology & Medicine. 47,1304-1309, 2009 Akari Suzuki, Atsushi Taira, Takayuki Ogi, Abstracts of the 138th Annual Meeting of the Pharmaceutical Society of Japan, 2018 Naoyuki, et al. , Journal of functional Foods. 29, 19-28, 2017

The present invention is an Nrf2 activator and heme ossigenase 1 (HO) having an oxidative stress reducing effect in the fields of cosmetics, pharmaceuticals, and foods containing a pyranocoumarin compound containing a button bow fu (long-lived grass, sakuna) extract as an active ingredient. -1) To provide a whitening / skin-beautifying agent having an inhibitory effect on tyrosinase activity and melanin production by a heme metabolite associated with the activity.

Table 1 lists the abbreviations of the Nrf2 activator of the pyranocoumarin compound (formula 1) isolated from the Siler (long-lived grass, Sakuna) extract.

The present invention provides a whitening / skin-beautifying agent by inhibiting tyrosinase activity and suppressing melanin production by an Nrf2 activator containing a pyranocoumarin compound containing a button bofu (long-lived grass, sakuna) extract as an active ingredient. The gist of the present invention is as follows.

As a result of diligent studies to solve the above problems, the present inventor has achieved inhibition of tyrosinase activity and suppression of melanin production by reducing oxidative stress of Nrf2 activator of pyranocumalin compound containing freckle extract and. It was found that it can whiten and beautify the skin by reducing stains and freckles, and can prevent skin cancer melanoma due to excessive melanin accumulation. The mechanism of action is due to the effects of inhibition of tyrosinase activity and inhibition of melanin production by heme metabolites associated with the activity of antioxidant protein HO-1 associated with Nrf2 activation, and all Nrf2 activators have similar effects. Have.

That is, in the mode of the present invention, the following Nrf2 activators having an oxidative stress reducing effect inhibit tyrosinase activity and suppress melanin production to provide sun-induced pigmented spots, chloasma preventive agents and melanoma preventive agents. That is.
1. 1. A whitening / skin-whitening agent containing an Nrf2 activator as an active ingredient by inhibiting tyrosinase activity and suppressing melanin production.
2. 2. 2. Cosmetics, pharmaceuticals and food raw materials by plants containing an inhibitor of tyrosinase activity and an inhibitor of melanin production containing an Nrf2 activator as an active ingredient. In addition, the tyrosinase activity inhibitory and melanin production inhibitor is applied externally to the skin or orally administered to any of the above-mentioned sun-induced pigmented spots and chloasma, which has a preventive effect on stains and freckles. And provide medicines that can prevent freckles.

As for the pyranocoumarin compound (formula 1) contained in the button bow fu extract used in the present invention, all plants containing the agent in addition to button bow fu are used as raw materials for the extract. Moreover, the raw material itself can be used as a cosmetic raw material or a food material. The melanin production inhibitor by the Nrf2 activator in the present invention is contained in one or more of cosmetic raw materials, food materials and pharmaceuticals, and is an aqueous solution, an emulsion, a water dispersion, a paste, a gel or a powder in the form of a dried product. It may be in the form of granules, a regular form, or the like. In addition, cosmetics and foods containing a whitening / skin-whitening agent by activating Nrf2 can be produced by a known processing method.

Examples Although examples of the present invention will be described below, the present invention is not limited to the following examples.

Cell culture B16F1 melanosite cells were cultured in an incubator (5% CO ₂ , 37 ° C.) using DMEM medium (containing 10% fetal bovine serum, 100 U / ml penicillin and 100 μg / ml streptomycin).

Melanin production suppression test B16F1 melanocytes are subcultured in a CO ₂ incubator (5% CO ₂ , 37 ° C.) using DMEM medium (10% fetal bovine serum, 100 U / ml penicillin, 100 μg / ml streptomycin). In the melanin production suppression test, cells were sprinkled 1 ml each on a 12-well plate (1 × 10 ⁶ cells / well) and cultured in a CO ₂ incubator (5% CO ₂ , 37 ° C.) for 24 hours. After exchanging the culture supernatant, a sample was added to the cultured cells and cultured for 24 hours. The cultured cells are exfoliated by tripricin treatment, the culture supernatant is removed after centrifugation, and the cells are centrifuged twice with PBS to obtain cell pellets. The cell pellet is air-dried for 1 day and then melanin is extracted with 1N NaOH. The extract is transferred to a 96-well microplate and the absorbance at a wavelength of 450 nm is measured using a microplate reader. At this time, if the absorption wavelength was affected by the color of the sample, the measurement was performed after centrifugation and washing with an organic solvent suitable for the sample in a timely manner. The melanin production inhibition rate (%) of each sample was calculated from the melanin production inhibition rate of the sample-added cells (B) with respect to 100% of the control of the sample-unadded cells (A) (Equation (1)).
FIG. 1 shows the activity of suppressing melanin production by the Button Siler extract. Melanin production was suppressed in a concentration-dependent manner. In addition, the pterixin of the pyranocoumarin compound (Table 1), which is the Nrf2 active substance contained therein, also showed suppression of melanin production (Fig. 2). The Nrf2 activator expresses the antioxidant protein HO-1. As a result, the activity of suppressing melanin production by the heme metabolite (bilirubin) associated with the HO-1 activity was confirmed (Fig. 3).

Tyrosinase Inhibitory Activity Test To evaluate the tyrosinase activity inhibition of a sample, use a McIlvaine buffer (pH 6.8, 100 μl) test solution containing 0.3 mg / ml L-tyrosine solution (100 μl), sample (2 μl) and purified water (88 μl). , Preincubation was performed at 37 ° C. for 10 minutes. Tyrosinase (1530 U / ml, 10 μl) was added to this test solution, and the mixture was reacted at 37 ° C. for 30 minutes. After the reaction, the absorbance (A ₁ ) at a wavelength of 450 nm was measured with a microplate reader. In the test, the same treatment and measurement were performed for L-tyrosine-free (A ₂ ), sample-free (B ₁ ), and sample and L-tyrosine-free (B ₂ ), and the tyrosinase activity of the sample was determined from the formula (1). The inhibition rate (%) was calculated.
FIG. 4 shows the results of inhibition of tyrosinase activity by the Button Siler extract. The tyrosinase activity was suppressed in a concentration-dependent manner. In addition, bilirubin, a heme metabolite produced by the expression of HO-1 associated with the activity of the Nrf2 active substance (Table 1), suppressed the tyrosinase activity activated over time (Fig. 5).

Expression test of HO-1 protein B16F1 melanocytes (5.0 × 10 ⁵ cells / ml) were precultured for 24 hours, and the test compound (final concentration (μM) 25, 50, 100) selected from Table 1 was added. Then, it was cultured at 24:00. Each protein was separated from the collected cultured cells by SDS-PAGE method, and HO-1 and β-Actin (housekeeping protein for expression control) were detected by Western blot analysis. FIG. 6A shows the results of Western blotting of HO-1 protein expression of Nrf2 activator (pterixin). Each expression level (HO-1 / β-Actin) was calculated as the expression rate with respect to 100% of the test compound untreated (control) (FIG. 6 (b)). From this result, the Nrf2 activator in melanocytes expressed HO-1 in a concentration-dependent manner. From the above results, it can be seen that the Button Siler extract and the Nrf2 activator contained therein (Table 1) suppress melanin production associated with reduction of oxidative stress by expressing the antioxidant protein HO-1. Shown. On the other hand, kojic acid, which is known as a melanin production inhibitor, does not express HO-1, indicating that it is different from the melanin production inhibitory mechanism of the Nrf2 activator.

It shows the activity of suppressing melanin production by the button bow extract. It shows the activity of suppressing melanin production by Nrf2 activator (pterixin). It shows the activity of suppressing melanin production by the heme metabolite (bilirubin) of HO-1. Inhibition of tyrosinase activity by Siler extract is shown. Inhibition of tyrosinase activity by Nrf2 activator (pterixin) is shown. The HO-1 protein expression (a) by the Nrf2 activator (pterixin) and the expression activity rate (b) thereof are shown.

According to the present invention, if an Nrf2 activator such as a freckle extract and a melanomaline compound as an active ingredient thereof is used as an active ingredient in the cosmetics field, the pharmaceutical field, the general food field and the functional food field, ultraviolet rays and the like can be used. It can be used as a whitening / skin-beautifying agent that can prevent skin disorders caused by oxidative stress and prevent liver spots such as spots and freckles and sun-induced pigmented liver spots, and as a preventive agent for melanoma (melanoma).

Claims (6)

Whitening and skin-beautifying agent by inhibiting tyrosinase activity of Nrf2 activator and suppressing melanin production Whitening and skin-beautifying effects by inhibiting tyrosinase activity and suppressing melanin production by Nrf2 activator of Button Siler (long-lived grass, Sakuna) extract (R ¹ in the formula is CH ₃ CO group, R ² is CH ₃ CH = C (CH ₃ ) CO group (cis form, trans form), (CH ₃ ) ₂ C = CHCO group, (CH ₃ ) ₂ CH -CH ₂ CO group, CH ₃ CH _2- CH (CH ₃ ) CO group derivative, or R ¹ CH ₃ CH _2- CH (CH ₃ ) CO group and R ² CH ₃ CO group compound) Inhibition of tyrosinase activity and inhibition of melanin production by heme metabolites such as biberdin and bilirubin by heme ossigenase 1 (HO-1) associated with Nrf2 activity Liver spots and freckles due to abnormal melanin pigmentation caused by inhibition of tyrosinase activity by the Nrf2 activator according to any one of claims 1, 2, 3 and 4 and hem metabolites such as biverzine and bilirubin and suppression of melanin production. Preventive agents for spots, sun-induced pigmented spots and melanoma (melanoma) The Nrf2 according to any one of 2 to 3 or a whitening / skin-beautifying agent by inhibiting tyrosinase activity by the Nrf2 activator according to claim 1 and suppressing melanin production, wherein the Nrf2 activator is administered externally or orally to the skin. Cosmetics, pharmaceuticals or foods containing an activator, the Nrf2 activator according to claims 1 to 5, and the heme metabolites biberdin and bilirubin associated with the activity.