JP2007031315A - Transcription factor nrf2 activator and skin care preparation, cosmetic, and food and drink formulated with the transcription factor nrf2 activator - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a Nrf2 activator enhancing antioxidant biophylaxis mechanism against oxidation stress by activating Nrf2 which is a transcription factor protein inducing gene expression of an enzyme group relating to antioxidant biophylaxis mechanism of living organism against oxidation stress, and a skin care preparation, cosmetic, and food and drink formulated with the same, especially an activator which activates Nrf2 in consideration of the fact that the Nrf2 existing in cytoplasm transfers to the nuclei by growth of the oxidation stress and accumulates to activate transcription in expression process of the antioxidizing enzyme group and to provide a Nrf2 activator and a skin care preparation and cosmetic formulated with the activator. <P>SOLUTION: The transcription facter Nrf2 activator comprises at least on of the extract of vegetables belonging to genus Pratia, genus Citrus or Curcuma domestica. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a transcription factor Nrf2 activator, a skin external preparation containing the transcription factor Nrf2 activator, a cosmetic, and a food and drink, and more specifically, an enzyme involved in an antioxidant defense mechanism of a living body against oxidative stress Nrf2 activator that can enhance antioxidant defense mechanism against oxidative stress by activating Nrf2 which is one of transcription factor proteins that induce gene expression of the group, and skin containing the Nrf2 activator The present invention relates to external preparations, cosmetics, and food and drink.

  In recent years, reports suggesting a relationship between oxidative stress and aging and various pathological conditions have been reported. Oxidative stress is a phenomenon accompanied by a decrease in biological functions due to the generation of active species such as free radicals. Lipids and sugars cause chain oxidation reactions to produce peroxides and secondary products, and protein denaturation. And the loss of enzyme activity, DNA strand scission and base modification are recognized as free radical cytotoxicity mechanisms.

  Living organisms originally have an antioxidant defense mechanism, and themselves produce various antioxidants and enzyme groups to combat oxidative stress. However, when the amount of oxidative stress exceeds the allowable range of the antioxidant defense mechanism, or when the antioxidant defense ability due to aging decreases, the balance between oxidation and antioxidant breakdown. Such a breakdown of the balance between oxidation and antioxidant is considered to be one of the factors that promote aging and develop or exacerbate diseases such as arteriosclerosis, cancer, and heart disease.

  A typical example of the promotion of aging is the aging phenomenon of the skin, and examples thereof include wrinkle formation and reduced elasticity, pigmentation, loss of texture, and reduced moisturizing function. These skin aging phenomena are exacerbated by oxidative stress caused by UV exposure. In addition to the above-mentioned arteriosclerosis, cancer, heart disease, etc., oxidative stress is associated with the onset and worsening of the disease state, as well as hypertension, cataract, Parkinson's disease / Alzheimer, gastric ulcer, liver disease, renal failure, etc. Is also thought to cause oxidative stress. For example, atherosclerosis is said to be caused by oxidized LDL, and antioxidants are used for this treatment. Furthermore, ischemia reperfusion injury, tobacco, alcohol, etc. are also recognized as causing oxidative stress and exacerbating diseases associated with oxidative stress disorders.

  For oxidative stress as described above, vitamin C and its derivatives, ubiquinol, vitamin E, polyphenol and other antioxidants, and antioxidants are generally used as health supplementary foods for preventing aging and preventing disease. A method of ingesting a plant extract containing an agent is employed. Moreover, these antioxidants and plant extracts are also used as external preparations for the purpose of preventing and improving skin spots, wrinkles and the like.

However, some of these antioxidants are prone to oxidation and have low stability in the preparation, and there are cases where the effect after external use or after ingestion is poor and sufficient effects may not be obtained. . In addition, there are a wide variety of sites and types of stress that are subjected to oxidative stress, and it cannot be said that sufficient effects can be obtained only by external application or ingestion of a specific antioxidant.
That is, the conventional antioxidants and plant extracts as described above are only transient ones used individually for each symptom that develops as a result of the occurrence of oxidative stress. It has not been developed from the essential point of view of how to maintain the oxidation defense mechanism.

  On the other hand, it is known that various antioxidant enzymes are expressed in the living body against oxidative stress as described above. For example, heme oxygenase (HO-1) and peroxide redoxin (MSP23) are oxidized. It is known that it contributes to the elimination of peroxides and radicals generated during stress.

In recent years, it has been reported that various genes are involved as transcription factors in the transcriptional stage leading to the expression of the enzyme group in the gene expression induction for expressing the antioxidant enzyme group as described above. Nrf2 is known as one of such transcription factors.
For example, in the specification [0006] of Patent Document 1 below, the inventors of the patent application have reported for the first time in the world that “Nrf2 is a transcriptional activator of the second phase enzyme group of the foreign body metabolism system”. Is described.

  Furthermore, [0006] also reveals that “the second phase of the foreign body metabolic system is controlled by controlling the nuclear translocation of Nrf2 having the strongest transcriptional activity. In other words, the Keap1-Nrf2 functional complex The following molecular mechanisms have been proposed: Nrf2 is bound to the propeller domain of Keap1 located in the cytoplasm under non-stress conditions, but is metabolized by oxidative stress or phase 1 detoxification enzymes When a cellular regulatory response to such a stimulus occurs, Nrf2 is released from Keap1 and quickly translocates to the nucleus, so that Nrf2 forms a heterodimer with another bZip partner such as small Maf. As a heterodimer is expressed in the target gene via an antioxidant responsive element present in the gene regulatory region of the target gene. Eg to have a second phase detoxification enzymes and oxidative stress-reducing protein) is described as. "To transactivation.

  As is clear from these descriptions, the transcription factor Nrf2 is present in the cytoplasm when oxidative stress is not occurring, and when oxidative stress occurs, it moves from the cytoplasm to the nucleus and accumulates in the nucleus and accumulates as described above. In addition, it activates transcription in the expression process of detoxification enzymes and oxidative stress-reducing proteins.

JP 2003-18943 A

  As described above, the present invention is characterized in that Nrf2 present in the cytoplasm is transferred to and accumulated in the nucleus due to the occurrence of oxidative stress, and activates transcription in the expression process of antioxidant enzymes and the like. In view of the above, it is an object to provide an activator that activates such Nrf2, and a skin external preparation, a cosmetic, and a food and drink containing such an activator.

The present inventors pay attention to the action of Nrf2 in the antioxidant defense mechanism of the living body as described above,
It has been found that the extract of the genus Platya, the extract of the citrus genus plant, and the turmeric extract have an activation action based on the nuclear accumulation of Nrf2 as described above, and have the function of strengthening the antioxidant defense mechanism. The invention has been completed.

  That is, the invention according to claim 1 relating to the transcription factor Nrf2 activator contains at least one of an extract of a platia genus plant, an extract of a citrus genus plant, or an extract of a turmeric. The invention according to claim 2 is the transcription factor Nrf2 activator according to claim 1, wherein the extract of the plant of the genus Platya is an extract of primrose, an extract of platia puberla, or an extract of platia anglata. It is characterized by being. Furthermore, the invention according to claim 3 is that in the transcription factor Nrf2 activator according to claim 1, the extract of the citrus genus plant is a chimpi extract, seihi extract, or kissi extract of the citrus genus plant. Features.

  Furthermore, the invention described in claim 4 relating to the external preparation for skin is characterized in that the transcription factor Nrf2 activator described in any one of claims 1 to 3 is blended. The invention according to claim 5 is characterized in that, in the external preparation for skin according to claim 4, an anti-aging component is blended in addition to the transcription factor Nrf2 activator.

  Furthermore, the invention according to claim 6 relating to cosmetics is characterized in that the transcription factor Nrf2 activator according to any one of claims 1 to 3 is blended. The invention according to claim 7 is characterized in that, in the cosmetic according to claim 6, an anti-aging component is blended in addition to the transcription factor Nrf2 activator.

  Furthermore, invention of Claim 8 which concerns on food-drinks mix | blended the transcription factor Nrf2 activator in any one of Claims 1 thru | or 3. The invention according to claim 9 is characterized in that, in the food or drink according to claim 8, an anti-aging component is blended in addition to the transcription factor Nrf2 activator. Furthermore, the invention according to claim 10 relating to the glutathione production promoter is characterized by containing at least one of an extract of a platia genus plant, an extract of a citrus genus plant, or a turmeric extract.

According to the present invention, an activator having an excellent activation action of the transcription factor Nrf2 can be provided, and by adding such an activator, high suppression of various symptoms caused by oxidative stress is achieved. It has become possible to provide external preparations for skin, cosmetics, and foods that are effective in preventing and improving skin aging and various diseases.
In addition, it is not easy to oxidize itself like conventional antioxidants, so it has superior stability and long-lasting effects compared to antioxidants. The effect of being able to maintain an antioxidant defense mechanism according to various types of stress, not depending on the site subjected to oxidative stress, and not transiently used individually for the symptoms of There is.

  As described above, the transcription factor Nrf2 activator of the present invention contains at least one of a Platya plant extract, a Citrus plant extract, or a Turmeric extract. The plant of the genus Platya is a plant belonging to the genus Platya of the family Oleaceae, and the kind thereof is not particularly limited in the present invention.

  Primrose (Pratia nummlaria) is a perennial plant native to southern China. Pratia puberula is a perennial plant native to Tasmania, and Pratia angulata is a perennial plant native to New Zealand.

  Citrus plants are plants belonging to the citrus family Citrus. In the present invention, an extract of citrus peel is mainly used. Specifically, for example, an extract of Chinpi, an extract of Sehi, and an extract of Tachibana (Kippi) are used. Chinpi is the ripe pericarp, Seich is the immature pericarp, and Kippi is the pericarp of citrus tachibana.

  An extract (extract) of a platia genus plant is prepared, for example, by extracting a leaf, stem, flower, bark, seed, fruit, root, etc. of a platia genus plant with an extraction solvent. In addition to these dried leaves, stems, flowers, bark, seeds, fruits, roots, etc., it is also possible to use, for example, those already marketed as tea. Moreover, the extract (extract) of a citrus genus plant extracts the dried material which dried the peel or immature fruit of the citrus genus plant with an extraction solvent. Turmeric (Curcuma longa L.) is a perennial plant belonging to the family Ginger, and can use whole plants such as leaves, stems, flowers, bark, seeds, fruits and roots, but it is desirable to use rhizomes. Moreover, the crude drug which dried the rhizome of turmeric can also be used.

  The method for preparing these extracts (extracts) is not particularly limited. For example, the extract is extracted at low temperature or room temperature using various appropriate solvents, or extracted under heating. As the extraction solvent, for example, water, lower monohydric alcohols such as methanol and ethanol, liquid polyhydric alcohols such as glycerin, propylene glycol, and 1,3-butylene glycol, hydrous alcohols, and the like can be used alone or in combination. . As an example of a preferable extraction method, there may be mentioned a method of performing extraction for 1 to 5 days at room temperature using ethanol or 1,3-butylene glycol having a water content of 0 to 100% by volume, followed by filtration.

  Since the transcription factor Nrf2 activator of the present invention contains at least one of the extract of Platya plant, the extract of Citrus plant, or the turmeric extract, the transcription factor Nrf2 activator is It may consist of any one of the genus plant extract, the Citrus plant extract, or the turmeric extract, or two or three of these three extracts are transcription factor Nrf2 activity It may be contained in the agent. For example, it is possible to include an extract of the genus Platya and an extract of the citrus genus plant in the transcription factor Nrf2 activator.

  Moreover, it is also possible to make the transcription factor Nrf2 activator contain an extract of two or more kinds of Platya plants or an extract of two or more kinds of Citrus plants. For example, when the transcription factor Nrf2 activator contains two or three of the extract of the primrose, the extract of Platya pverula, or the Platya angrata, or the chimpi extract of the citrus genus plant, the Sehi extract Or two or three of the extract of the Kippi extract are contained in the transcription factor Nrf2 activator.

  Furthermore, “containing” means that the transcription factor Nrf2 activator of the present invention consists of only one or more of Platya plant extract, Citrus plant extract, or Turmeric extract. It may mean that other than these components may be contained.

The above-mentioned transcription factor Nrf2 activator is blended in the external preparation for skin, cosmetics and food and drink of the present invention. The blending amount of the transcription factor Nrf2 activator is not particularly limited. For example, in the case of an external preparation for skin and cosmetics, the total amount of the external preparation may be 0.0005 to 5% by weight as a dry solid amount. desirable. If the amount is less than 0.0005% by weight, the activation of Nrf2 is not always exhibited sufficiently, and if the amount exceeds 5% by weight, the improvement corresponding to the increased amount is not always seen. is there.

  The topical skin preparation means various drugs applied to the skin, and its use is not limited, such as cosmetics, quasi-drugs, and pharmaceuticals. Therefore, the external preparation for skin of the present invention and the cosmetic of the present invention may overlap with each other. The form of the external preparation for skin is not particularly limited, and any type can be used as long as it is applicable to the skin such as liquid, ointment, cream, gel, and aerosol. When the external preparation for skin is a cosmetic, it can be used as a tonic, rinse, shampoo, astrigent, etc. for cosmetics such as emulsions, lotions, packs, cleaning agents, foundations, and scalp.

  In addition to the transcription factor Nrf2 activator, which is the essential component, depending on the form of the external preparation for skin, components used for external skin preparations such as normal cosmetics and external medicines, such as purified water, lower alcohol, polyhydric alcohol, oily Ingredients, powders, surfactants, thickeners, coloring materials, preservatives, humectants, fragrances and the like can be blended within a range that does not impair the effects of the present invention. When the cosmetic of the present invention is an oral cosmetic, it can be used as a toothpaste, gel, mouthwash, dispersion or the like.

  Furthermore, the food and drink in the present invention includes not only food and drink in a narrow sense but also so-called health foods and the like, beauty and health-oriented foods and drinks, and the like. The form of the food or drink is not particularly limited, and examples thereof include solid agents such as powders, tablets, capsules, granules and powders, solutions such as solutions, emulsions and suspensions, and freeze-dried agents. It can also be drunk as tea. Furthermore, as food, it can be used as a favorite food such as gum, candy, yogurt, ice cream, soft drink. Depending on the type of food or drink, purified water, physiological saline, dextrin, glucose, mannitol, sucrose, lactose, starch, amino acids, gelatin, albumin, fatty acid glycerides, polyethylene glycol, polyoxyethylene sorbitan fatty acid ester, hydroxyethylene starch Etc. can be used in the range which does not impair the effect of this invention.

  Since the transcription factor Nrf2 activator of the present invention has the actions and effects as described above, it can be applied to pharmaceuticals other than external preparations for skin, for example, pharmaceuticals such as oral preparations. When oral administration is applied to such internal medicine or health food as described above, the dosage is preferably 0.01 to 5000 mg per day in terms of the transcription factor Nrf2 activator.

  The transcription factor Nrf2 activator of the present invention is combined with an existing anti-aging component, and is incorporated into the above-mentioned external preparations for skin, cosmetics, foods and drinks, pharmaceuticals, etc., thereby causing various symptoms caused by oxidative stress. In addition to the original effect of the transcription factor Nrf2 activator that exerts a high inhibitory effect on the skin, an excellent effect for preventing and improving skin aging, various diseases and the like occurs.

  In this case, the anti-aging component can prevent various aging symptoms such as a component having an antioxidant effect, a component having a cell activating effect, a component having a melanin-inhibiting effect, and a component capable of preventing wrinkles. It means to include ingredients widely. And in this anti-aging component, the plant extract which contains such an anti-aging component as an active ingredient is also included. For example, ascorbic acid (vitamin C) and its derivatives and salts thereof, tocopherol (vitamin E) and its derivatives, niacinamide, niacin, glutathione, hypotaurine, thiotaurine, cysteine, N-acetylcysteine, cysteamine, etc. And derivatives thereof, retinol (vitamin A) and derivatives thereof, carotenoid components such as carotene and astaxanthin and derivatives thereof, hydroquinone and derivatives thereof, arbutin and derivatives thereof, plant extracts containing arbutin, coenzyme Q10, α-lipo Acid, Pycnogenol and plant extracts containing Pycnogenol, Polyphenols such as catechin, Flavonoids, Carotenoid, Tannin, Lignin, Saponin, Procyanidin, Rutin Contains hesperidin, quercetin, ferulic acid and ferulic acid-containing plant extracts, glycyrrhizic acid and its derivatives, glycyrrhetinic acid and its derivatives, panthenol and its derivatives, pantothenic acid, grabridine and its derivatives, soy isoflavone and soy isoflavone Examples include soybean extracts, ellagic acid and plant extracts containing ellagic acid, higher unsaturated fatty acids such as linoleic acid and linolenic acid and derivatives thereof, and natural fats and oils containing 10% or more of these higher unsaturated fatty acids.

  Ascorbic acid and its derivatives and salts thereof include ascorbic acid phosphates and salts such as water-soluble ascorbic acid derivatives such as sodium ascorbate phosphate, magnesium ascorbate phosphate, palmitoylated ascorbate phosphate and its salts Ascorbic acid derivatives such as ascorbyl tetraisopalmitate, ascorbyl palmitate, ascorbyl palmitate, ascorbyl palmitate, ascorbyl isopalmitate, ascorbyl stearate, ascorbyl diisopalmitate, etc. In addition to esters and the like, ascorbic acid polypeptides, ascorbic acid chitosan and the like can be mentioned.

  Examples of the vitamin E derivative (tocopherol derivative) include tocopherol nicotinate, tocopherol linoleate, tocopherol succinate, tocopherol acetate and the like. Examples of the retinol derivative include retinol palmitate, retinol linoleate, and retinol acetate.

  The transcription factor Nrf2 activated by the activator of the present invention controls the expression levels of various antioxidant enzyme groups, detoxification enzyme groups, etc. existing in the living body as described above. Nrf2 present in the cytoplasm moves to and accumulates in the nucleus due to oxidative stress such as active oxygen, and expresses an antioxidant enzyme group and a detoxification enzyme group.

  The antioxidant enzyme group and the detoxifying enzyme group whose expression levels are controlled by Nrf2 include glutathione S transferase, γ-glutamyl cystinyl synthetase, heme oxygenase 1, quinone oxide reductase 1, glutathione peroxidase, glutathione, thioredoxin, bilirubin, metallothionein, Examples include ferritin and peroxide redoxin. Many other gene products under the control of Nrf2 have been reported.

  Examples of the present invention will be described below.

Example 1
The primrose was dried and chopped, 100 ml of water containing 50% by volume of water, ethanol or the like was added, extracted at room temperature for 5 days, and then filtered to obtain a primrose extract. At this time, the dry solid content was 1.35% by weight.

(Example 2)
Platia Buberla was dried and chopped, 100 ml of water containing 50% by volume of water, ethanol, etc. was added, extracted at room temperature for 5 days, and then filtered to obtain Platya Buberla extract. At this time, the dry solid content was 1.45% by weight.

(Example 3)
Platia angrata was dried and chopped, 100 ml of water containing 50% by volume of water, ethanol, etc. was added, extracted at room temperature for 5 days, and then filtered to obtain a platia anglatha extract. At this time, the dry solid content was 1.25% by weight.

Example 4
The skin of Unshu mandarin orange was dried and chopped, 100 ml of water containing 50% by volume of water, ethanol, etc. was added, extracted at room temperature for 5 days, and then filtered to obtain a chimpi extract. At this time, the dry solid content was 1.45% by weight.

(Example 5)
The unripe fruit of Satsuma mandarin was dried and chopped, and 100 ml of water, ethanol, etc. having a water content of 50% by volume was added thereto, followed by extraction at room temperature for 5 days, followed by filtration to obtain a Sehi extract. At this time, the dry solid content was 1.65% by weight.

(Example 6)
Tachibana peel was dried and chopped, 100 ml of water containing 50% by volume of water, ethanol, etc. was added, extracted at room temperature for 5 days and filtered to obtain a Kippi extract. At this time, the dry solid content was 1.25% by weight.

(Example 7)
Turmeric rhizomes were dried and chopped, 100 ml of water containing 50% by volume of water, ethanol, etc. was added, extracted at room temperature for 5 days, and then filtered to obtain a turmeric extract. At this time, the dry solid content was 1.75% by weight.

(Test Example 1)
[Intracellular glutathione level measurement test]
It has been reported that a substance having a transcription factor Nrf2 activating action increases intracellular glutathione concentration. Therefore, the transcription factor Nrf2 activator of the present invention can be confirmed by activating the transcription factor Nrf2 activator by measuring the intracellular glutathione concentration by conducting the following test.

Normal human neonatal foreskin-derived fibroblasts were used for the test. Cells are suspended in Dulbecco's modified Eagle's medium (Sigma) containing 10% by volume fetal calf serum (ICN), seeded in a 60 mm diameter petri dish, and CO ₂ incubator (95% by volume air, 5% by volume carbon dioxide). ), And cultured at 37 ° C. for 24 hours.

After culturing for 24 hours, the sample extract medium was replaced with a dry solid content of the plant extract contained in the medium at a concentration of 100 μg / mL as a dry residue. That is, the culture medium cultured as described above is removed from the petri dish, and Dulbecco containing no fetal calf serum added in advance so that the dry residue of the extract of primrose (Example 1) is contained at a concentration of 100 μg / mL. The modified Eagle medium (manufactured by Sigma) was replaced with a petri dish and cultured in a CO ₂ incubator (95 vol% air, 5 vol% carbon dioxide) at 37 ° C for 24 hours.

After culturing for 24 hours in this way, the amount of glutathione was measured using a total glutathione amount measuring kit (manufactured by Dojin) according to the same instructions. That is, a culture of fibroblasts (5 × 10 ⁵ cells) as described above was centrifuged at 200 g for 10 minutes at 4 ° C., the supernatant was discarded, 300 μL of PBS was added, and again at 200 g for 10 minutes at 4 ° C. Washed by centrifuging and discarding the supernatant. 80 μL of 10 mM HCl was added to the washed culture, and freezing and lysis were repeated twice to disrupt the cell membrane. Furthermore, 20 μL of 5% sulfosalicylic acid was added and centrifuged at 8000 g for 10 minutes, and the supernatant was used as a measurement sample. The glutathione concentration was measured on a 96-well microplate (96 well plate). First, 140 μL of Coenzyme working solution and 20 μL of Enzyme working solution included in the kit were placed in each well and incubated at 30 ° C. for 5 minutes.

  Next, 20 μL of the measurement sample prepared as described above and GSH standard solution for preparing a calibration curve attached to the kit were added to each well and incubated at 30 ° C. for 10 minutes. Furthermore, 20 μL of Substrate working solution was added to each well, incubated at room temperature for 10 minutes, and the absorbance of each well was measured with a microplate reader (Dainippon Pharmaceutical Co., Ltd.). The glutathione concentration was determined by the reaction of 5,5′-dithiobis-2-nitrobenzoic acid [5,5′-dithiobis-2-nitrobenzoic acid] with 5-mercapto-2-nitrobenzoic acid [5-mercapto- Utilizing the reduction to 2-nitrobenzoic acid], the absorbance of this substance was measured at 412 nm, and calculated from a calibration curve prepared with a glutathione standard set at the same time. The amount of protein in the cell lysate obtained as described above was determined using a protein assay (Bio-Rad). This cell lysate contained glutathione extracted from human fibroblasts, and the amount of intracellular glutathione was expressed as the amount of glutathione per protein amount.

  Extracts of plants other than primrose (Example 1), that is, extracts of Pratia puberla (Example 2), extracts of Platia anglata (Example 3), chimpi extract (Example 4), seihi extract As for (Example 5) and the Kippi extract (Example 6), the dry residue is also contained at a concentration of 100 μg / mL in Dulbecco's modified Eagle medium (manufactured by Sigma) that does not contain fetal bovine serum. And the amount of glutathione as described above was measured. In addition, the density | concentration of the sample addition medium in this case can be suitably changed in the range in which a human fibroblast does not die.

  On the other hand, the amount of glutathione was also measured in the same manner in an additive-free medium to which these plant extracts were not added (Dulbecco's modified Eagle medium without fetal calf serum). Then, in addition to the amount of glutathione in human fibroblasts, the value (ratio) when no additive was taken as 100 was also calculated. The test results are shown in Table 1.

  As is clear from Table 1, the amount of glutathione when no primrose extract, platia pverula extract, platia angrata extract, chimpi extract, seihi extract, and kippi extract is added is not added. In this case, the amount was about 1.4 times or more, and the glutathione synthesis promoting effect was observed. In particular, in the case of the extract of primrose, the effect of promoting the synthesis of glutathione was excellent, that is, about 1.8 times or more that when the amount of glutathione was not added.

A similar test was performed using normal human neonatal foreskin keratinocytes. However, the medium was replaced with Dulbecco's modified Eagle medium, and serum-free liquid medium Humedia-KG2 (manufactured by Kurabo Industries) for normal human epidermal keratinocyte proliferation was used. The medium contains insulin, hEGF (human epidermal growth factor), hydrocortisone necessary for the growth of epidermal keratinocytes, and gentamicin and amphotericin B as antibacterial agents. The test results are shown in Table 2.

  As is apparent from Table 2, the amount of glutathione when primrose extract, platia pverula extract, platia angrata extract, chimpi extract, seihi extract and kissi extract are added is not added. The effect of promoting the synthesis of glutathione was observed more than in the case. In particular, in the case of the extract of primrose and the extract of chimpi, the effect of promoting the synthesis of glutathione was excellent, about 1.4 times or more that when the amount of glutathione was not added.

(Test Example 2)
[Confirmation of transcription factor Nrf2 activation]
Normal human neonatal foreskin skin-derived fibroblasts were used. Cells were suspended in Dulbecco's modified Eagle's medium (Sigma) containing 10% fetal bovine serum (ICN), seeded in 100 milliliters, and placed in a CO ₂ incubator (95 vol% air, 5 vol% carbon dioxide), 37 Culturing was performed under the condition of ° C. After 24 hours, the medium was replaced with a sample-added medium supplemented with primrose extract (Example 1), and nucleoprotein (protein present in the cell nucleus) was extracted after 6 hours of culture.

The sample addition concentration was 100 μg / mL as the concentration at which the effectiveness was obtained in the test example, that is, the dry residue of the medium. The nucleoprotein solution was prepared by the following procedure. The cells were suspended in Lysis buffer A (20 mM HEPES (pH 7.6), 20 volume% glycerin, 10 mM NaCl, 1.5 mM MgCl ₂ 0.2 mM EDTA, 1 mM DTT, 0.1 volume% NP-40) and placed on ice. After incubation for 20 minutes, centrifugation was performed at 1500 ° C. for 5 minutes at 4 ° C., and the resulting precipitate was added to Lysis buffer B (20 mM HEPES (pH 7.6), 20 vol% glycerin, 500 mM NaCl, 1.5 mM MgCl ₂ , 0. 2 mM EDTA, 1 mM DTT, 0.1 vol% NP-40) was added, and the mixture was stirred at 4 ° C. for 20 minutes, and centrifuged again at 4 ° C., 1500 g for 10 minutes to obtain a supernatant.

  The nucleoprotein solution was dissolved in Sample buffer (125 mM Tris-HCl pH 6.8, 4 wt% SDS, 20 vol% glycerol, 10 vol% mercaptoethanol) and subjected to electrophoresis. The protein on the gel was transferred to a nitrocellulose membrane (manufactured by Bio-Rad) and immersed in TBS buffer (20 mM Tris.base, 137 mM NaCl) containing 3% nonfat dry milk for 1 hour for blocking. After washing with a TBS buffer containing 0.05% by volume of Tween 20, the TBS buffer containing a primary antibody Nrf2 antibody (manufactured by Santa Cruz) was replaced and immersed overnight, and then a secondary antibody HRP-labeled antibody (Santa It was immersed for 1 hour in TBS buffer containing (Cruise).

After washing with a TBS buffer containing 0.05% by volume of Tween 20, the HRP substrate (manufactured by Santa Cruz) was reacted with the nitrocellulose membrane and the film was exposed to the results obtained by densitometry (manufactured by Atto). Analyzed. As an internal standard, Lamin B antibody (manufactured by Santa Cruz), which is always present quantitatively in the nucleus in the primary antibody, was used in the same manner to determine the amount of Nrf2 relative to the internal standard.
Extracts of plants other than primroses (Example 1), that is, extracts of Platya pverula (Example 2), extracts of Platya angrata (Example 3), and chimpi extract (Example 4) The same operation was performed for the Sehi extract (Example 5) and the Kippi extract (Example 6) in the same manner. On the other hand, the same operation was performed for the non-added medium to which these plant extracts were not added. Table 3 shows the ratio of the amount of Nrf2 in each sample when each plant extract was added with the amount of Nrf2 with respect to the internal standard without addition being 1.

  As is clear from Table 3, the amount of Nrf2 was remarkably higher when primrose extract, platia pulvera extract, platia angrata extract, chimpi extract, seihi extract and kippi extract were added, Therefore, these extracts were found to induce and activate Nrf2 nuclear accumulation. In particular, when primrose extract was added, the amount of Nrf2 exceeded 6 times that without addition.

(Test Example 3)
[Wrinkle improvement and whitening effect test]
As described above, primrose extract, which is one of primrose extract, platia pulvera extract, platia angrata extract, chimpi extract, seihi extract and kippi extract having Nrf2 activating action was formulated. A cream was prepared by the following method (Prescription Example 1), and wrinkle improvement and whitening effect were examined. A cream containing ascorbic acid magnesium phosphate, which is an ascorbic acid derivative generally used as an antioxidant, which is one of the above-mentioned anti-aging components, is the one without added primrose extract. As Comparative Example 1, a cream containing Coenzyme Q10 was compared as Comparative Example 2. Further, in order to confirm the combined effect of Nrf2 activator and antioxidant, in addition to the extract of primrose extract, the above ascorbic acid magnesium phosphate salt was formulated (Formulation Example 2), and the coenzyme Q10 was formulated (Prescription Example 3) was also tested.

  Squalene, cetyl isooctanoate, and microcrystalline wax are heated and dissolved, and then clay mineral and POE glycerol triisostearic acid ester (surfactant) are added and kept at 70 ° C. to uniformly disperse and dissolve to obtain an oily gel It was.

  Next, the primrose extract was dissolved in purified water to a predetermined concentration, heated to 70 ° C., and then slowly added to the oily gel with sufficient stirring. After uniformly mixing with a homomixer, deaeration and cooling to 30 ° C. gave a cream. The composition and blending ratio of the obtained cream of Test Example 3 are as follows.

(Prescription Example 1)
Composition ratio (wt%)
Squalene 20%
Cetyl isooctanoate 8.5%
Microcrystalline wax 1%
Clay mineral 1.3%
POE glycerol triisostearate 0.2%
Primrose extract 1%
Water remaining

(Prescription example 2)
Composition ratio (wt%)
Squalene 20%
Cetyl isooctanoate 8.5%
Microcrystalline wax 1%
Clay mineral 1.3%
POE glycerol triisostearate 0.2%
Primrose extract 1%
Magnesium ascorbate phosphate 2%
Water remaining

(Prescription Example 3)
Composition ratio (wt%)
Squalene 20%
Cetyl isooctanoate 8.5%
Microcrystalline wax 1%
Clay mineral 1.3%
POE glycerol triisostearate 0.2%
Primrose extract 2%
Coenzyme Q10 0.03%
Water remaining

(Comparative Example 1)
Composition ratio (wt%)
Squalene 20%
Cetyl isooctanoate 8.5%
Microcrystalline wax 1%
Clay mineral 1.3%
POE glycerol triisostearate 0.2%
Magnesium ascorbate phosphate 2%
Water remaining

(Comparative Example 2)
Composition ratio (wt%)
Squalene 20%
Cetyl isooctanoate 8.5%
Microcrystalline wax 1%
Clay mineral 1.3%
POE glycerol triisostearate 0.2%
Coenzyme Q10 0.03%
Water remaining

(Comparative Example 3)
Composition ratio (wt%)
Squalene 20%
Cetyl isooctanoate 8.5%
Microcrystalline wax 1%
Clay mineral 1.3%
POE glycerol triisostearate 0.2%
Water remaining

〔Test method〕
Subjects selected 90 women with wrinkles, spots and / or dullness from women aged 25 to 54 years. A panel of 15 persons per test cream of Formulation Examples 1 to 3 and Comparative Examples 1 to 3 was applied to the face after washing the face twice daily in the morning and night for 3 months. The wrinkle improvement effect by application was evaluated according to the following criteria. The degree of improvement of wrinkles was visually determined according to the following evaluation criteria, and the elasticity was measured using a cut meter with elasticity as an index.

  A cute meter is a device that measures the amount of change and temporal change in the skin that is sucked when a probe is brought into close contact with the skin and the inside of the probe is made negative pressure. The amount of displacement 5 seconds after depressurization is defined as UF, and the amount of displacement immediately after the depressurization is released is defined as Ur. The value of the cream of each of the prescription examples 1 to 3 and the comparative examples 1 and 2 was expressed as a ratio by setting the value obtained by subtracting the Ur / UF before the start of the test from the Ur / UF after the cream application test of the comparative example 3 to 100. In addition, when arbitrary five places of each test subject were measured and Ur / UF was averaged, there was no test subject whose value before the test exceeded that after the test. The higher the value, the higher the elasticity than before the start of the test.

The brightness of skin color and the visibility of spots and stains were visually determined according to the following evaluation criteria, and at the same time, the brightness of skin color was measured with a color difference meter (manufactured by Minolta). The evaluation results were obtained by subtracting the L ^* value before the start of the test from the L ^* value after the test of the cream of Comparative Example 3 as 100, and the L ^* values of the creams of each of Prescription Examples 1 to 3 and Comparative Examples 1 and 2 The rise was expressed as a ratio. In addition, when arbitrary 5 places were measured for each subject and the average of the L ^* values was calculated, there was no subject whose value before the test exceeded that after the test. The higher the value, the higher the brightness and the brighter skin color.

(Evaluation criteria)
<Wrinkle evaluation><Contents>
Effective elasticity and gloss were given, and wrinkles were improved.
Slightly effective elasticity and luster were given, but it did not improve wrinkles.
Invalid No change before use.
<Whitening evaluation><Contents>
Effective The skin tone became brighter, and spots and dullness became inconspicuous.
Slightly effective The skin tone became brighter, but the spots and dullness remained unchanged.
Invalid No change before use.

  The results of the wrinkle improvement evaluation are shown in Table 4, and the whitening evaluation is shown in Table 5.

As is clear from Table 4, the cream of Formulation Example 1 formulated with the extract of primrose improves wrinkles and tarmi compared to the cream of Comparative Example 3 that does not contain the extract of primrose and antioxidants. It became clear that it can become a certain skin and beautiful skin.
And even if it compared with the cream of the comparative examples 1 and 2 containing the antioxidant generally considered to be effective, an excellent result was obtained rather. Furthermore, it was found that the prescription examples 2 and 3 containing both primrose and an antioxidant enhance the wrinkle improving effect, and in particular, the prescription example 3 containing coenzyme Q10 together with primrose gives very excellent results. It was.

  Further, as is clear from Table 5, the cream of Formulation Example 1 containing the extract of Primrose can obtain a whitening effect as compared with the cream of Comparative Example 3 that does not contain Primrose extract and an antioxidant, and is beautiful. It became clear that it was skin. And even if it compared with the cream of the comparative examples 1 and 2 containing the antioxidant generally considered to be effective, an excellent result was obtained rather. Further, it was found that the whitening effect was further enhanced in Formulation Examples 2 and 3 containing both primrose and antioxidant.

(sensory evaluation)
Subjects were asked about the effects on the skin when using the creams of Formulation Examples 1 to 3 and Comparative Examples 1 to 3. Tables 6 and 7 show the results of the responses. Table 6 shows the results of satisfaction with wrinkles, and Table 7 shows the results of satisfaction with spots.

  As is clear from Tables 6 and 7, a good effect was confirmed for the cream of Formulation Example 1 in which the extract of primrose was blended by sensory evaluation indicating satisfaction with wrinkles and spots. Further, the creams of Formulation Examples 2 and 3 combined with an antioxidant were found to have a better effect.

  From the determination results of Tables 4, 5, 6, and 7 above, the cream blended with the extract of primrose that is one of the Nrf2 activators of the present invention is excellent in safety when applied to the skin, and is firm and glossy. It was also found to be effective in improving wrinkles and improving spots and dullness. Moreover, the effect was further strengthened by using an antioxidant together.

(Test Example 4)
[Prevention of lipid peroxide]
The intake of primrose extract and the effect on blood lipid peroxide level were investigated. A commercial feed (manufactured by CLEA Japan, CE-2 powder form) was mixed with 3% by weight extract of primrose and allowed to eat freely in 10-week-old Wistar rats (8 per group), and after 8 weeks in blood Lipid peroxide levels and glutathione peroxidase (GPX) activity were measured. Blood was collected from the tail vein of rats, and heparin was added at a concentration of 10 U / mL as an anticoagulant.

  For the lipid peroxide concentration, a kit of lipid peroxide test Wako (manufactured by Wako Pure Chemical Industries, Ltd.) was used. This was a method for obtaining the lipid peroxide concentration in the sample as the malondialdehyde concentration, and the measurement was performed according to the same instructions. Briefly, physiological saline is added to the blood, N / 12 sulfuric acid and 10% phosphotungstic acid aqueous solution are added to the supernatant obtained after centrifugation, and distilled water and TBA are added to the precipitate obtained by centrifugation again. Reagent (8.8 mol / L acetic acid, 3.35 mg / mL 2-thiobarbituric acid) was added and heated in a boiling water bath for 60 minutes.

  After cooling, extraction with n-butanol was performed, and the fluorescence of the upper layer was measured after centrifugation. For the measurement, a microplate reader (Dainippon Pharmaceutical Co., Ltd.) equipped with a filter having an excitation wavelength of 515 nm and a fluorescence wavelength of 553 nm was used. The lipid peroxide concentration was calculated from a standard curve prepared by simultaneously measuring a standard solution (5 nmol / ml 1,1,3,3-tetraethoxypropane and malondialdehyde given quantitatively) of this kit.

  Next, GPX activity was changed to BIOXYTECH pl.GPx. It measured using the kit of 340TM (made by OXIS). GPX has the function of detoxifying peroxide using reduced glutathione. In this kit, oxidized glutathione produced by this reaction is reduced by glutathione reductase in the presence of NADPH, and the amount of NADPH that decreases at this time is determined from the absorbance (340 nm). The experimental procedure followed the instructions of the kit.

  That is, centrifugation was performed at 1500 g for 10 minutes at 4 ° C., the plasma and leukocyte fractions in the supernatant were removed, and red blood cells were collected. The collected red blood cells were diluted 4 times with distilled water and used as a measurement sample. Next, this measurement sample was mixed with 350 μL of Assay buffer attached to the kit and 350 μL of attached NADPH solution, and the absorbance at 340 nm was measured using a spectrophotometer (manufactured by Beckman Coulter). Furthermore, 350 μL of t-butyl hydroperoxide solution attached to the kit was added and mixed well, and then the absorbance at 340 nm was measured again. After 3 minutes, the absorbance at 340 nm was measured again.

  The hemoglobin concentration was measured to correct for GPx activity per hemoglobin. Hemoglobin produces extremely stable cyanmethemoglobin by the action of potassium ferrocyanide and potassium cyanide, and the concentration of the product is proportional to the absorbance at 540 nm using a spectrophotometer. Since the content of this product and hemoglobin is in direct proportion, the hemoglobin concentration in the sample can be calculated from a standard curve that can be created from the absorbance of a standard hemoglobin product. That is, 10 μL of blood or hemoglobin standard product and 2.5 mL of reaction solution (0.00014 wt% sodium bicarbonate, 0.02 wt% potassium ferricyanide, 0.005 wt% potassium cyanide) were mixed and allowed to stand for 15 minutes, and the absorbance at 540 nm. Was measured.

  The test results are shown in Table 8.

  As is clear from Table 8, it was found that the intake of primrose extract significantly decreased the amount of lipid peroxide compared to the non-intake group, and the GPx activity increased. This result suggests that intake of primrose can prevent oxidation in the body and prevent symptoms and diseases that exacerbate oxidative stress.

Claims (10)

  A transcription factor Nrf2 activator comprising at least one of an extract of a Platya plant, an extract of a Citrus plant, or a turmeric extract.   The transcription factor Nrf2 activator according to claim 1, wherein the extract of the plant of the genus Platya is an extract of primrose, an extract of Platya pverula, or an extract of Platya anglata.   The transcription factor Nrf2 activator according to claim 1, wherein the Citrus plant extract is a Citrus plant chimpi extract, Sehi extract, or Kippi extract.   An external preparation for skin, comprising the transcription factor Nrf2 activator according to any one of claims 1 to 3.   The skin external preparation according to claim 4, wherein an anti-aging component is blended in addition to the transcription factor Nrf2 activator.   A cosmetic comprising the transcription factor Nrf2 activator according to any one of claims 1 to 3.   The cosmetic according to claim 6, wherein an anti-aging component is blended in addition to the transcription factor Nrf2 activator.   A food or drink comprising the transcription factor Nrf2 activator according to any one of claims 1 to 3.   The food or drink according to claim 8, wherein an anti-aging component is blended in addition to the transcription factor Nrf2 activator.   A glutathione production promoter characterized by containing at least one of an extract of a platia genus plant, an extract of a citrus genus plant, or a turmeric extract.