KR101347442B1 - Singlet oxygen scavenger, anti-skin-aging agent, anti-wrinkle agent, anti-sagging agent, agent for improving moisture content in the skin, skin-whitening agent, melanin formation inhibitor, nitrogen monooxide scavenger and antioxidant, each utilizing helipyrone A - Google Patents

Abstract

The present invention finds a new action on Helipyrone A, the single-use oxygen scavenger, skin aging enhancer, wrinkle improver, sag improver, skin moisture amount containing the effective use of Helipyrone A (Helipyrone A) An object of the present invention is to provide an improving agent, a whitening agent, a melanin inhibitor, a nitric oxide scavenger or an antioxidant.

Description

Singlet oxygen scavenger, anti-skin-aging agent, anti singlet oxygen scavenger, anti-skin-aging agent, skin aging improver, wrinkle improver, sag improver, skin moisture improver, whitening agent, melanin inhibitor, nitric oxide scavenger -wrinkle agent, anti-sagging agent, agent for improving moisture content in the skin, skin-whitening agent, melanin formation inhibitor, nitrogen monooxide scavenger and antioxidant, each utilizing helipyrone A}

The present invention relates to Helipyrone A.

Non-Patent Document 1 (Journal of ethnopharmacology 33 (1991) p51-55) discloses that helipyrone A represented by the general formula (1) has an antibacterial effect.

Non Patent Literature 1: Journal of ethnopharmacology 33 (1991) p51-55

Non-Patent Document 2: Esahak Ali, et al., Phytochemistry, 1982, 21, 243-244

DISCLOSURE OF INVENTION

Problems to be solved by the invention

An object of the present invention is to discover a novel action on Helicyrone A and to promote its effective use.

Means for solving the problem

The main configuration of the present invention is as follows.

1. Singlet oxygen scavengers, skin aging enhancers, wrinkle improvers, sag improvers, skin moisture improvers, whitening agents, melanin inhibitors, nitric oxide scavengers or antioxidants containing Helicyrone A represented by the formula (1).

2. An oral composition containing any one of 1.

3. The composition for external use of skin containing any one of 1.

4. Food containing any one of 1. agent.

5. A medicament containing any one of 1.

6. Cosmetics containing any one of 1. agents.

Effects of the Invention

With respect to Helipyrone A, the effects of singlet oxygen scavenging, skin aging, wrinkle improvement, sag improvement, skin moisture content improvement, whitening, melanin inhibition, nitric oxide scavenging or anti-oxidation were confirmed. Various novel agents, medicines, foods, and cosmetics utilizing this can be provided.

1 is a graph showing wrinkle score.

2 is a graph showing skin moisture content.

3 is a graph showing skin hardness.

4 is a graph showing the amount of carbonylated protein (relative value).

5 is a graph showing the amount (relative value) of 8-hydroxy 2'-deoxy guanosine in skin tissue.

6 is a graph showing the mass of peroxide in skin tissue.

7 is a graph showing nitrogen monoxide scavenging ability by the ESR method.

8 is a graph showing nitrogen monoxide scavenging ability by the colorimetric method (Griess method).

9 is a graph showing the peroxynitrite scavenging ability by the colorimetric method.

10 is a graph showing the inhibitory ability to increase Proliferation Cell Nuclear Antigen (PCNA).

11 is a graph showing the loricrin inhibitory ability.

12 is a graph showing keratin 1 inhibitory activity.

Carrying out the invention  Best form for

Helipyrone A is represented by the following formula (1).

[Formula 1]

Helipyrone A has been found to have novel effects such as anti-wrinkle effect, skin aging improvement, photo aging, whitening, melanin production inhibition, antioxidant, singlet oxygen scavenging and nitric oxide scavenging. Based on these novel knowledge, it can use as a cosmetic composition, a food addition material, and a pharmaceutical material.

As cosmetics and foods, anti-wrinkle effect, skin aging improvement, photo aging, whitening, melanin production inhibition, antioxidant, singlet oxygen scavenging can be expected, skin aging improving agent, photoaging inhibitor, whitening agent, melanin production inhibitor, antioxidant, singlet oxygen It can be used as a scavenger and a nitrogen monoxide scavenger.

Examples of the cosmetics (including quasi-drugs) containing helipyrone A of the present invention include moisturizing cosmetics for face or hands, feet and body, such as lotions, emulsions, creams, gels, and multilayer cosmetics. have. Moreover, it can also be used as hair cosmetics, such as makeup cosmetics, such as a lipstick and a foundation, washing | cleaning agents, such as a face wash, a hand cleaner, and a body shampoo, a shampoo, a rinse, a treatment, and a hair growth agent.

As the cosmetics (including quasi-drugs) containing helipyrone A of the present invention, a material generally used as a cosmetic composition can be used in addition to helipyrone A. For example, hydrocarbon oils, ester oils, fatty acids, higher alcohols, emulsions such as silicones, sterols, ceramides, polyhydric alcohols, sugars, humectants such as pyrrolidone carboxylic acids, amino acids, betaines, and nonionics Surfactants, anionic surfactants, cationic surfactants, amphoteric surfactants, surfactants such as semipolar surfactants, lecithin, polymer emulsifiers, water-soluble polymers, thickeners such as dextrin palmitate, inorganic powders, organic powders, antibacterial agents, bactericides, A pH adjuster, a chelating agent, a coloring agent, a fragrance, a plant extract, etc. can be mix | blended.

As food containing Helicyrone A of this invention, Helicyrone A can be used as a food as it is, or various nutrients can be added, and you may mix | blend with the food of interest. For example, starch, lactose, maltose, plant oil powder, cacao butter powder, stearic acid and the like are added, and then, by using conventional means, a form suitable for edible food such as granular, granular, tablet, It can be molded into capsules, pastes, etc. to form health foods, health functional foods, and the like. It may be added to various foods, for example, meat processed foods such as ham and sausage, fish processed foods such as fish paste and skewered fish paste, bread, confectionery, butter, powdered milk and fermented milk products, and water, fruit juice, milk and soft drinks. You may use it in addition to drink.

As a medicament containing Helipyrone A of the present invention, it is mixed with a solid or liquid pharmaceutical non-toxic carrier suitable for administration methods such as oral administration, transdermal administration, rectal administration, injection, and the like. It may be administered in the form of a pharmaceutical preparation.

Such preparations include, for example, solid preparations such as tablets, granules, powders and capsules, liquid preparations such as solutions, suspensions and emulsions, lyophilized preparations, and the like. It can be prepared by.

Examples of the non-toxic carrier for medical use include glucose, lactose, sucrose, starch, mannitol, dextrin, fatty acid glycerides, polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid esters, amino acids, gelatin, albumin, Water, physiological saline, and the like. Moreover, it is also possible to add conventional additives suitably, such as a stabilizer, a wetting agent, an emulsifier, a binder, and an isotonic agent as needed.

[Chemical Synthesis of Helipyrone A]

The chemical synthesis of Helipyrone A is disclosed in Non Patent Literature 2 (Esahak Ali, et al., Phytochemistry, 1982, 21, 243-244).

For example, in this invention, it synthesize | combined as follows.

172 g of Compound 1 diethylketone and Compound 2 tetrahydro-1,4-oxazine (morpholine) in the presence of titanium tetrachloride using 400 mL of hexane solvent 1,000 g was reacted for 3 hours at 4 ° C. and purified by distillation to give Compound 3 (N-isopropenyl) -tetrahydro-1,4-oxazine ((N-isopropenyl) -tetrahydro-1,4-oxazine). 503.57 g (yield 81.1%).

Using 200 mL of toluene solvent, 167 g of Compound 3 (N-isopropenyl) -tetrahydro-1,4-oxazine was added to Compound 4 ethyl mal. 81 g of ethyl malonyl chloride was reacted with methanol-ice cooling (-17 ° C. to 11 ° C.) to synthesize Compound 5 represented by Chemical Formula 2.

Reaction of compound 3 and compound 4 was complete | finished by addition of 2N hydrochloric acid, and the magnesium sulfate drying process was performed after chloroform extraction. Next, 200 mL of toluene and 400 mL of 25% hydrochloric acid were sequentially added to the reaction solution, and the toluene layer was recovered by liquid-liquid distribution. The toluene layer was washed with 400 mL of 0.1 N hydrochloric acid, and after drying the magnesium sulfate, toluene was removed by a vacuum evaporator to obtain an orange oily solution. 1 kg of PPA (polyphosphoric acid) was added to this solution, and it cyclized at 110 degreeC-118 degreeC, and compound 5 was synthesize | combined. Compound 5 was cooled to room temperature, recovered by chloroform extraction, and the magnesium sulfate was dried to remove chloroform by vacuum evaporation. Silica gel chromatography (chloroform: methanol = 50: 1 (v / v)) gave 45.7 g of orange solid compound 5 as a solid. The yield of this reaction was 18.3%.

Compound 6 Helicyrone A was chemically synthesized by polymerizing formaldehyde with 1 N hydrochloric acid. 45.7 g of compound 5 was dissolved in 460 mL of ethanol, and 247.81 g of formaldehyde (37%) was refluxed at 79 ° C in the presence of 2.5 mL of concentrated hydrochloric acid to precipitate a crystal. The crystals were washed with 300 mL of ethanol to obtain 34.01 g of Helipyrone A as white crystals. The yield of this reaction was 71.6%.

The physical-property value of the obtained helipyrone A is shown below.

Appearance: White Crystal

NMR spectrum (400 MHz, solvent; CDCl ₃ )

Molecular weight: 320.341 g / mol (mass spectrometry)

Molecular Formula: C ₁₇ H ₂₀ O ₆ (Mass Spectrometry)

Melting Point: 218-220 ℃

Antioxidant Evaluation

Helipyrone A was evaluated for singlet oxygen scavenging action. The evaluation method was performed by the ESR spin trapping method using 2,2,6,6-tetramethyl-4-piperidinol (2,2,6,6-tetramethyl-4-piperidinol (4-OH TEMP)) as a spin trapping agent. Measured. First, 0.1 mL of 40% (v / v) N, N-dimethylformamide solution of Helicyrone A prepared at an arbitrary concentration was added to a 96-well microplate having a flat bottom, and then distilled water 0.02 mL was added and mixed with 0.04 mL of 100 mM 4-OH TEMP aqueous solution and 0.04 mL of 0.1 mM rose bengal aqueous solution to prepare a mixed solution of 0.2 mL. As a light-sensitizing light source, a single-light oxygen was generated by irradiating a light emitting diode (green LED) having a maximum wavelength of 530 nm for 20 minutes from the back of the microplate. This mixed solution was transferred to the flat aqueous solution cell for ESR, and the ESR spectrum was measured. The singlet oxygen scavenging ratio was determined from the ESR spectral intensity, and plotted against the concentration to obtain a 50% inhibition concentration IC50 value. In addition, as a comparison, conventional polyphenol compounds (silbin, 3,4-dihydroxyphenylethanol, (-)-catechin, (-)-epicatechin gallate, quercetin, rosemarine acid, carnosic acid ), Caffeic acid, kojic acid, and β-carotene) were obtained at the same time. The results are shown in Table 1.

As is clear from these results, it was recognized that helipyrone A eliminated singlet oxygen at lower concentrations compared to other polyphenol compounds. In other words, it was clear that Helipyrone A showed excellent singlet oxygen scavenging action.

[Melanin Production Inhibition Test]

Commercially available 6-well plates of mouse B16 melanoma cells were each incubated to confluent using DMEM containing 10% FBS (fetal bovine serum) in culture under conditions of 5% CO ₂ , 37 ° C. 100 nM αMSH as a melanogenesis stimulator was added, followed by incubation for 72 hours with the addition of 0.0001% of helipyron A. Solvent conditions were 10% FBS containing DMEM medium, 0.2% acetonitrile was contained in all wells in consideration of the solubility of helipyrone A. After completion of the culture, the cell fractions were collected, the melanin pigment was dissolved by 0.1 N NaOH alkali treatment, and the absorbance (ABS) at 405 nm was measured.

Melamine production inhibition

Melamine production inhibition rate [%] = 100 × {1- (ABS [Sample]) / (ABS [Control])}

Control: DMEM medium containing 0.2% acetonitrile

Sample: 0.201% acetonitrile containing 0.0001% helipyrone A (DMEM medium)

.

As a result, 22.1% ± 8.4 (S.D.) melanin production was inhibited when 0.0001% of helipyron A was added.

[Wrinkle suppression test by ultraviolet irradiation]

Nude mice (hairless mice) that form wrinkles and sags, which are a phenomenon of skin aging by continuously irradiating weak ultraviolet rays for a long time, were conducted as animal models, and the effects of the administration of helipyrone A in combination with feed were analyzed.

I. Experimental Animal

Species, line, sex

Species: Mice, Line: Hos: HR1 (nude mouse), Gender: Female

Age of breeding start

Breeding from 5 weeks of age, at the same time mixed with the feed was started to administer.

II. Breeding environment

Set temperature and humidity: 24 ± 1 ℃, relative humidity 55 ± 5%

Air Conditioning Equipment: All Fresh

Illumination time: 12 hours auto on / off

Breeding equipment: 5 plastic cages / cage

Feed: Free intake of powdered sterilized feed MF-1 (Oriental Yeast Industry Co., Ltd.) or 0.01% (w / w), 0.05% (w / w), 0.1% (w) of helipyron A in MF-1, respectively. / w) or β-carotene with excellent antioxidant effect was mixed 0.01% (w / w), 0.05% (w / w).

III. Exam Schedule

UV irradiation conditions: UV-A wave 14 J / cm 2 (irradiation time about 1 minute), UV-B wave 20 mJ / cm 2 (irradiation time about 50 minutes) using an ultraviolet lamp for 10 weeks every other day (every day) Was investigated without applying restraint stress. The total irradiation amount for 10 weeks (35 times) is UV-A wave 490 J / cm 2 and UV-B wave 700 mJ / cm 2.

In the group which continued ultraviolet irradiation, the last ultraviolet irradiation was performed 24 hours before dissection. Fasting was performed 18 hours before dissection. The skin moisture content was measured using a simple moisture meter (Moisture Checker ^™ , scalase) by a non-invasive method before dissection.

Nembutal anesthesia was performed and the appearance was photographed. And a replica of skin distribution was taken.

By dissection, hepatic extraction, whole blood recovery, backing, and abdominal skin were recovered.

Organs were rapidly cryopreserved and whole blood was centrifuged (3,000 G, 20 min, room temperature) to recover plasma and cryopreserved. The skin was cryopreserved except that it fixed a certain area of the belly and was used for tissue staining.

The appearance of Bissett DL et al. (Photochem Photobiol. (1987) Vol.46, No.3, pp367-), focusing on the appearance of the sagging of the neck and the formation of wrinkles on the back. pp378). That is, it quantified according to the definition shown below, and the sample which was difficult to judge added 0.5 as an intermediate value. The data of the wrinkle score is graphed and shown in FIG. 1.

Score 0

The occurrence of wrinkles is not recognized at all.

In a stationary state, the line perpendicular to the spine direction is not recognized, or slightly accepted, and depending on the individual's movement, the line is lost.

Score 1

The occurrence of minor wrinkles is clearly recognized.

A line perpendicular to the spine direction is recognized, and the line is lost as the individual moves.

Score 2

The occurrence of moderate wrinkles is clearly recognized.

The line is clearly recognized for the direction of the spine, and as the individual moves, the line is not lost and is permanent.

Score 3

The occurrence of deep wrinkles is clearly recognized.

The line is clearly recognized for the direction of the spine, the shadow is recognized for the line, and according to the individual's movement, the line is not lost and is permanent.

Wrinkle score was 0.05% of MF-1 group 0.1 which blended 0.1% of helipyron A for 10 weeks of ultraviolet irradiation, helipyron A for 10 weeks of ultraviolet irradiation compared with that of MF-1 (control) group 2.4 for 10 weeks of ultraviolet irradiation. Formation of wrinkles was remarkably suppressed as MF-1 type | group group 0.6 which mix | blended 0.01% of MF-1 type | formula which was mix | blended and 0.01% of helipyron A for 10 weeks of ultraviolet irradiation. The wrinkle score of the MF-1 (control) diet group which elapsed for 10 weeks without irradiating an ultraviolet-ray was 0.2, and generation | occurrence | production of the wrinkle by ultraviolet irradiation is suppressed completely by ingesting 0.05-0.1% of compound formula of helipyron A.

From the results of the skin findings, remarkable anti-wrinkle effect of helipyrone A is recognized. The administration of helipyrone A to the mouse is about 520 with a free feed of 0.1% of the daily dose of human, using 4 g of the daily dose of the mouse and the surface area equation y = (3√x) 2. It is an amount equivalent to about 52 mg / day / 60 kg body weight with a mg / day / 60 kg body weight and 0.01% free of mixed feed, so that it can be easily consumed as food.

In addition, the β-carotene feeding group having antioxidant activity did not show an anti-wrinkle effect.

The wrinkle score of the MF-1 diet group which contained 0.05% of β-carotene for 10 weeks of ultraviolet irradiation was 2.0, and the MF-1 group group wrinkle score which contained 0.01% of β-carotene for 10 weeks of ultraviolet irradiation was 2.3.

[Inhibition of Skin Drying with UV Irradiation]

Immediately before dissection, the skin moisture content was measured using a non-invasive method using a Moisture Checker ^™ (scalase), and compared with each group for the average measured value measured five times per animal. The measurement result of skin moisture content is shown in FIG.

In the MF-1 group of the 10-week UV irradiation + Helicyrone A combination, the skin moisture content decreased in the MF-1 (control) diet group for 10 weeks of ultraviolet irradiation and became dry (skin content 31.4%). The skin moisture content was 41.3% in the 0.1% blend, the skin moisture content was 40.2% in the 0.05% blend, and 38.2% in the 0.01% blend. The skin moisture was maintained around 40%. The skin moisture content of the helipyrone A formula group was higher than the skin moisture content of 35.5% of the MF-1 (control) diet group which had elapsed for 10 weeks without irradiating ultraviolet rays.

[Inhibition of Skin Curing with UV Irradiation]

Using a spring hardness tester (JIS Type C) skin viscoelasticity measuring device (Vesmeter: E-100S / Wave Cyber), measure the area 3 cm 2 cm toward the head and 0.5 cm to the right from the lumbar spine The average of hardness was calculated | required. The measurement result of skin hardness is shown in FIG.

It is generally known that the hardness known as an indicator of skin viscoelasticity is that skin aging progresses and the elasticity of the skin decreases as the numerical value is higher.

In the MF-1 type group of UV irradiation for 10 weeks + Helicyron A combination for 10 weeks of ultraviolet irradiation + MF-1 (control) diet group, the hardness of the hardness in 0.1% formulation of helipyron A 8.1, 0.05% blending hardness of 9.1, 0.01% blending hardness of 9.1, the degree to which the curing proceeds slightly compared to the hardness of 7.2 of the MF-1 (control) formula that elapsed for 10 weeks without irradiating ultraviolet rays. Curing was suppressed.

[Inhibition of Carbonylated Protein Production in Skin Tissue with UV Irradiation]

Carbonylated protein, one of the oxidized proteins, was evaluated using Oxyblot ^™ (CHEMICON). The concrete method is as follows. The skin of the mouse was weighed about 200 mg, homogenized by adding 0.2 ml of Lysis buffer (pH = 7.4) containing a Protease inhibitor cocktail at 4 ° C., and centrifuged for 12,000 × 20 minutes. The protein concentration was measured according to the method of Bradford using the filter which filtered the supernatant. The carbonyl group was DNPH for 20 μg of each protein sample. Proteins were separated by SDS-PAGE and transferred to PVDF membrane using a protein transcription device. The membrane after transcription is blocked in a PBS (-) solution containing 5% skim milk for 30 minutes at room temperature, and the skim milk is washed with PBS (-), and then reacted with anti-DNPH antibody at 4 ° C overnight, followed by biotinylation. It was reacted with anti mouse IgG for 1 hour. After washing, the PVDF film was exposed using a fluorescence detection kit (ECL PLUS), and the image was transferred to a medical automatic developing device. The results are shown in FIG.

As a result, the relative value of the amount of carbonylated protein in the 10-week UV irradiation + MF-1 (control) diet group is 259.4, while 0.1% of helipyron A in the MF-1 diet group of the 10-hour UV irradiation + Helicyrone A combination The relative value of the amount of carbonylated protein in the formulation was 25.3, the relative value of the amount of carbonylated protein in the 0.05% formulation was 35.6, and the relative value of the amount of carbonylated protein in the 0.01% formulation was 57.7. This was suppressed to 1/10-1/5. The relative value of the carbonylated protein content of the MF-1 (control) diet group after 2 weeks without irradiating ultraviolet rays is 23.7, and the relative value of the carbonylated protein amount of the helipyrone A 0.1% formula group is 25.7, which is the same degree. It was. Therefore, helipyrone A significantly suppresses the production of carbonylated protein associated with ultraviolet irradiation.

Antioxidant β-carotene feeding group was not recognized the inhibitory effect of the production of carbonylated protein.

The carbonylated protein of the MF-1 group that contained 0.05% of β-carotene for 10 weeks of ultraviolet irradiation was 153.1, and the carbonylated of the MF-1 group that contained 0.01% of β-carotene for 10 weeks of UV irradiation. The relative value of protein amount was 190.7.

[Inhibition of Production of 8-hydroxy-2'-deoxyguanosine (8-OH dG) in Skin Tissue with UV Irradiation]

8-OH dG was measured using immunohistochemical techniques. At the time of dissection of the animal, 1 cm 2 of skin 2 cm from the caudal part of the skin tissue distribution to the head and 0.5 cm from the lumbar spine to the right was cut out, anchored and paraffin embedded to preserve the skin tissue. Sections having a thickness of 3 μm were appropriately prepared, and deparaffinization and hydrophilization were performed based on a known method. Antigen activation was microwaved for 5 minutes in 0.01 M citric acid buffer (pH = 6.0). After cooling to room temperature, the mixture was reacted for 20 minutes in methanol containing 0.3% hydrogen peroxide at room temperature to inhibit endogenous peroxidase. After washing with water and washing 10 mM PBS (-), microwave treatment was performed for 5 minutes in a rabbit serum 75-fold 10 mM PBS (-) dilution solution to block blocking and dropping serum to form a primary antibody (N 45.1: manufactured by Nikken-Sale Co., Ltd.). ) Was reacted by microwave treatment at 5 μg / ml for 20 minutes. After washing twice with 10 mM PBS (-) and diluting the biotinylated secondary antibody (Biotinylated Rabbit Immunoglobulin M; DAKO) 300-fold, the antibody was reacted by microwave treatment for 5 minutes. After washing twice with 10 mM PBS (-), ABC reagent (ABC-HRP; manufactured by Vectastain) was reacted by microwave treatment for 5 minutes. The reaction was carried out at room temperature for 3 minutes 30 seconds using DAB (3,3-diaminobenzidine tetrahydrochloride: manufactured by DAKO) as a color developing reagent. After washing with water, dehydration and encapsulation were performed based on a known method. The staining state of the skin tissue was observed under a microscope, the image was read using Adobe Photoshop, and the staining part in a certain area of the image was digitized by NIH Imaging. The results are shown in Fig.

As a result, the relative value of 8-OH dG in the 10-week UV irradiation + MF-1 (control) diet group was 5.89, whereas 0.1% of helipyron A in the MF-1 diet group of the UV irradiation 10-week + Helicyron A combination The relative value of 8-OH dG is 0.87, and the relative value of 8-OH dG is 0.91 at 0.05%, and the relative value of 8-OH dG is 1.10 at 0.01%. It was suppressed from 15/100 to 19/100. Compared with the relative value of 1.00 of the amount of 8-OH dG of the MF-1 (control) formula group which had elapsed for 10 weeks without irradiating ultraviolet rays, the amount of 8-OH dG of the group of the 0.05% UV-Helipyron A formula and the 0.1% formula The relative value of is even lower. Therefore, helipyrone A significantly suppresses the generation of 8-OH dG accompanying ultraviolet irradiation.

In addition, the β-carotene feeding group having antioxidant activity did not show the inhibitory effect of 8-OH dG production.

The relative value of 8-OH dG amount of the MF-1 group which contained 0.05% of β-carotene for 10 weeks of ultraviolet irradiation was 5.2 and 8-OH of the MF-1 group which contained 0.01% of β-carotene for 10 weeks of ultraviolet irradiation. The relative value of the amount of dG was 6.3.

[Inhibition of Production of Lipid Peroxide in Skin Tissue Associated with Ultraviolet Irradiation]

TBARS (thiobarbituric acid reaction product, nmol / g wet weight) in skin tissue was measured by fluorescence analysis. Approximately 200 mg of the wet weight of each tissue was weighed, homogenated with 1 ml of 1.15% KCl, 4 ml of 1/12 N sulfuric acid and 0.5 ml of 10% tungsten phosphate were added sequentially, followed by centrifugation at 3,000 rpm for 10 minutes. It was done. The supernatant was removed, TBA reagent (thiobarbituric acid / acetic acid buffer) was added, and it heated and reacted for 1 hour in a boiling water bath. TBA reaction was performed with the sample which added 1,1,3,3- tetraethoxy propane separately for the determination of TBARS, and the analytical curve was produced. The results are shown in Fig.

After the completion of the heating reaction, the mixture was returned to room temperature by ice-cooling, 5 ml of n-butanol was added thereto, stirred, and centrifuged at 3,000 rpm for 10 minutes to recover the upper n-butanol layer, followed by fluorescence analysis (Ex 515 nm). , Em 553 nm). The fluorescence intensity meter used HITACHI F-2000.

As a result, in the MF-1 meal group of 10 weeks of ultraviolet irradiation + Helicyron A combination, 0.1% of helipyron A in the 10-hour UV irradiation + MF-1 (control) diet group compared to 50.8 nmol / mg of the lipid peroxide in the tissue. 30.9 nmol / mg of lipid peroxide in the tissue in the formulation, 0.05% lipid peroxide in the tissue in the 0.05% formulation, and 44.8 nmol / mg of lipid peroxide in the tissue in the 0.01% formulation. / 10 to 9/10 was suppressed. The mass of peroxide in the 0.1% and 0.05% group of the 10% helipadron A group and the 0.05% group of the UV irradiation group contained 30.2 nmol / mg of peroxide in the tissues of the MF-1 (control) group after 10 weeks without irradiation with UV. It is about the same. Therefore, helipyrone A significantly suppresses the generation of lipid peroxide accompanying ultraviolet irradiation.

In addition, there was little effect of inhibiting the production of lipid peroxide of the β-carotene feeding group having antioxidant activity.

The mass peroxide of the MF-1 group with 0.05% of β-carotene for 10 weeks of UV irradiation was 46.9 nmol / mg, and the mass peroxide of the MF-1 group with 0.01% of β-carotene for 10 weeks of UV irradiation was 51.1 nmol / mg.

[Evaluation of Nitric Oxide Scavenging Capacity by ESR Method]

Nitric oxide scavenging activity of (-) catechin (manufactured by Sigma), known as helipyron A and antioxidant, for 100 μM SNAP (S-nitroso-N-acetyl-D, L-penicillamine, dopant) Was compared.

The spin trapping agent (MGD) ₂ -Fe ² prepared by mixing N-methyl-D-glucamine-dithiocarbamate (hereinafter referred to as MGD, purchased from Labotech Co., Ltd.) and an aqueous solution of iron (II) sulfate. ⁺ Complex was used. This complex reacts with nitrogen monoxide to form a complex of (MGD) ₂ -Fe ^{2 +} -NO to generate an ESR spectral intensity, thereby measuring the ESR spectrum at each concentration of the nitrogen monoxide scavenger. The scavenging ability of was measured.

50 mM iron sulfate (II) 2 hydrate solvent: 100 μL of distilled water (final concentration 5 mM), 50 mM MGD solvent PBS (pH 7.4) 100 μL (final concentration 5 mM), test solvent: 10% acetonitrile 100 μL 699 μL of 100 mM phosphate buffer, 1 μL of 100 mM SNAP solvent 0.1 M sodium hydroxide (final concentration 100 μM) were added sequentially to a 1.5 mL volume of eppendorf tube and at 25 ° C. for 10 minutes from the time of SNAP addition. The reaction was stirred.

In the reaction of 25 ℃ 10 bungan is nitric oxide is released from ^{_{SNAP, (MGD) 2 -Fe 2}} + -NO signal intensity ratio = 1: was composed of 3 peaks of 1: 1. Nitric oxide scavenging ability was evaluated by how much this signal intensity increased or decreased with the test substance. Specifically, the nitrogen monoxide scavenging rate was obtained from the signal intensity (S) when the test substance was added based on the control signal intensity (C) of the control without adding the test substance, and is shown in FIG. 7. .

Nitrogen monoxide scavenging ratio = (1-S / C) * 100 (%)

As a result, helipyrone A improved nitric oxide scavenging rate in a concentration-dependent range from 25 μM to 200 μM (32.4% at 25 μM, 52.6% at 50 μM, 63.6% at 100 μM, 80.1% at 200 μM). Compared to the IC50 concentration of 51.4 μM, the nitrate scavenging rate was only 5.6% and 11.7% at 50 μM and 100 μM, respectively, and 31.4% at 250 μM and 47.4% at 500 μM. Was rate. IC50 concentration was calculated to be 547 μΜ.

The setting of the apparatus and apparatus used this time is shown below.

Instrument: JEOL JES TE200, X-band ESR device (100 KHz, Japan Electronics Corporation)

Set conditions for your device:

Center field: 335.0 ± 5.0 mT,

Microwave power: 4 kW,

Modulation amplitude: 0.1 mT,

Gain: 500,

Time constant: 0.1 sec,

Scanning time: 1 minute

Nitric Oxide Scavenging Capacity by Colorimetric Method (Griess Method)

Since nitrogen monoxide present in the solution is extremely unstable in an aqueous solution, in addition to the ESR method, the amount of nitrogen monoxide was indirectly evaluated by measuring nitrite ions, which are oxides of nitrogen monoxide. Known antioxidants (-) catechin, maltol and p-coumarinic acid were also evaluated and compared with helipyrone A. The measurement by the Griess method used the NO ₂ / NO ₃ Assay Kit-CII (Colorimetric) (propellant).

As the nitrogen monoxide generator, NOC-5 (1-hydroxy-2-oxo-3- (3-aminopropyl) -3-isopropyl-1-triazene) (the scavenger) is used until immediately before use. Cryopreservation was -20 ° C with an alkaline solution of 0.1 N sodium hydroxide. Nitrogen monoxide was generated by diluting 10,000 times with a buffer solution (pH = 7.6, hereinafter "buffer") in a kit and adjusting to a concentration of 100 µM. NOC-5, developed as a nitric oxide donor, is a labile compound (25 degrees, half life 25 minutes under pH 7.4) that is stable in alkaline solutions but produces nitrogen monoxide under neutral and acidic conditions.

To a 96-well plate with a flat bottom, 40 μL of a test solution (pH = 7.6) containing 1% acetonitrile was added at varying concentrations of the test substance. Thereafter, 20 µL of a buffer solution (pH = 7.6) was added, and 40 µL of a 100 µM NOC-5 solution was added and reacted at 25 ° C for 2 hours. After the reaction was completed, 50 μL of Griess reagent A in the kit was added and left to stand for 5 minutes. Then, 50 μL of the Griess reagent B was added thereto, and left for 10 minutes to conduct a color reaction. The absorbance at 540 nm was measured with a multiplate reader. The absorbance at the concentration of each test substance was S ₅₄₀ , the absorbance of the control without the test substance was C ₅₄₀ , and the absorbance of the blank without the test substance and NOC-5 was B ₅₄₀ . The nitrogen clearance rate (%) was computed and shown in Table 2 and FIG.

Nitric oxide scavenging rate (%) = 100 × (1- (S ₅₄₀ -B ₅₄₀ ) / (C ₅₄₀ -B ₅₄₀ )

The calibration curve was produced from the absorbance of the sample set in the concentration range of 0-100 μM of sodium nitrite solution instead of the NOC-5 solution, and the concentration of nitrite ion in the solution was determined. The nitrogen monoxide concentration generated from the NOC-5 solution can be obtained from the nitrite ion concentration. The intensity of nitric oxide scavenging ability was compared with the concentration (IC50, μM) which is 50% scavenging in this test system.

47.0 μM of nitrogen monoxide is produced as nitrite ion from 100 μM of NOC-5 solution. At this time, the decrease in absorbance due to the addition of the test substance, that is, the scavenging activity of nitrogen monoxide was recognized.

In the range of 50 μM to 400 μM of helipyrone A, nitric oxide scavenging ability was recognized and the IC50 concentration was 36.7 μM. On the other hand, the IC50 concentrations of (-) catechin, maltol and p-coumarinic acid were 190.0 µM, 94.0 µM, and 187.4 µM. Excellent nitric oxide scavenging ability of Helicyron A was confirmed.

From the above results, it is clear that helipyrone A of the present invention eliminates nitrogen monoxide, and it can be considered to be useful as a drug for the vascular system, the nervous system, and the immune system. This helipyron A eliminates excess nitrogen monoxide present in the living body, and is administered in advance when it is expected to act as a normal nitrogen monoxide concentration and excessive generation of nitrogen monoxide. It has a function to prevent it. In other words, in the present specification, the nitric oxide scavenging ability of the helipirone A does not mean completely erasing all of the nitrogen monoxide present in the living body, but is controlled so as to remove the excess nitrogen monoxide in the living body so as to be suitable for maintenance of the living body. I say that.

Nitrite Peroxide Scavenging Activity by Colorimetric Method

The nitrite peroxide scavenging ability of helipyrone A was measured by Griess method using NO ₂ / NO ₃ Assay Kit-CII (Colorimetric).

Known antioxidants (-) catechin, maltol and p-coumarinic acid were also evaluated and compared with helipyrone A.

To a 96-well plate with a flat bottom, 40 μL of a test solution (pH = 7.6) containing 1% acetonitrile was added at varying concentrations of the test substance. Thereafter, 10 μL of reductase and 10 μL of coenzyme in Kit were added to each well.

40 μL of SIN-1 (3- (4-morpholinyl) sidnonimine hydrochloride) was added as a nitrite peroxide generator and reacted at 37 ° C. for 2 hours. After the reaction was completed, 50 μL of Griess reagent A in the kit was added and left to stand for 5 minutes. Then, 50 μL of the Griess reagent B was added thereto, followed by 10 minutes of color reaction. The absorbance at 540 nm was measured with a multiplate reader. The absorbance of the test substance was S ₅₄₀ , the absorbance of the control without test substance was C ₅₄₀ , and the absorbance of the blank without test substance and SIN-1 was B ₅₄₀ . Was calculated and shown in Table 3, FIG.

Peroxide nitrite scavenging rate (%) = 100 × (1- (S ₅₄₀ -B ₅₄₀ ) / (C ₅₄₀ -B ₅₄₀ )

The calibration curve was produced from the absorbance of the sample set in the concentration range of 0-100 μM of sodium nitrate solution instead of the SIN-1 solution, and the concentration of nitrate ions in the solution was determined. From this concentration of nitrate ions, the concentration of nitrite peroxide generated from the SIN-1 solution can be obtained. The intensity of nitrite peroxide scavenging ability was compared with the concentration of 50% scavenging (IC50, μM) in this test system.

As a result, in the control, 8.18 μM of nitrite was generated from 50 μM of SIN-1, and for heliconon A of 24.04 μM with respect to an IC50 concentration of 50% scavenging of the nitrite, maltol 31.84 μM and p-coumarin The acid became 31.49 μM and (-) catechin 705.6 μM, showing that Helipyron A showed excellent nitrite peroxide scavenging ability.

Nitrite peroxide occurs in the reaction of nitrogen monoxide and superoxide anion in vivo, causing strong oxidation, damage to the body, inflammation. Helipyron A, which exhibits nitrite peroxide scavenging activity, functions as an antioxidant to suppress inflammation in a living body and to protect a biological component.

[Measurement of Tyrosinase Activity Inhibition]

100 μL of the test substance solution was added to a flat bottom 96-well plate, and 10 μL of a tyrosinase solution derived from 600 units / ml of mushroom was added thereto, followed by incubation at 30 ° C. for 10 minutes. Thereafter, 100 µL of a 6 mM L-DOPA (L-β- (3,4-dihydroxyphenyl) alanine) solution previously warmed to 30 ° C was added, and incubated with shaking at 30 ° C for 40 minutes. 100 mM succinic acid buffer (pH 5.5) containing 1% acetonitrile was used as the solvent of the test substance, and 100 mM succinic acid buffer (pH 5.5) was used as the solvent of tyrosinase and L-DOPA. After shaking, the absorbance was measured at the wavelength of 475 nm which is an index of melanin amount, and this value was set to S ₄₇₅ . In the case of no test substance added, control (C), the absorbance of C ₄₇₅ , L-DOPA-free sample blank (SB), the absorbance of SB ₄₇₅ , and sample and L-DOPA-free control blank (CB) And this absorbance was set to CB ₄₇₅ , and the tyrosinase inhibition rate was computed by following Formula. Arbutin (made by Tokyo Kasei Kogyo Co., Ltd.) was used as a positive control.

% Inhibition of tyrosinase activity = 100 × {1- (S ₄₇₅ -SB ₄₇₅ ) / (C ₄₇₅ -CB ₄₇₅ )}

As a result, the tyrosinase inhibition rate of helipyron A and arbutin at an addition concentration of 1 mM was 25.77% in arbutin, compared to 36.34% in helipyron A, so that helipyron A was better in tyrosinase than arbutin. It can be seen that heliopyron A has an inhibitory activity and exhibits a whitening effect.

[Tyrosinase Gene Expression Analysis in Mouse Skin]

When mice were fed Helipyron A, expression of the gene encoding tyrosinase was measured in mouse skin.

I. Experimental Animal

I-1. Species, strain, and gender: Species: mouse, strain: Hos: HR1 (nude mouse), Gender: female

I-2. Start of breeding week: 5 weeks of age (starting ultraviolet irradiation test after 6 weeks of breeding for 1 week)

I-3. Microbial Grade: SPF

I-4. Breather: Shimizu Experiment Materials Co., Ltd.

II. Breeding environment

Set temperature and humidity: 24 ± 1 ℃, relative humidity 55 ± 5%

Air Conditioning Equipment: All Fresh

Illumination time: 12 hours auto on / off

Breeding equipment: 5 plastic cages / cage

Feed: A powder sterilized feed MF-1 (Oriental Yeast Industries Co., Ltd.) was freely ingested, or a feed obtained by mixing 0.1 wt% of helipyron A with MF-1 was separately prepared.

Administration of Test Subjects: Free intake as a powdered blend. Fasted 18 hours before dissection.

Water supply: free intake of sterile tap water

III. Anatomy

Anesthesia was introduced by intraperitoneal administration of the anesthetic nembutal (40 mg / kg) after measuring the body weight and skin moisture of the fasted subject 18 hours before dissection.

The abdomen was dissected and heparin blood was collected from the heart. Next, after visual observation was performed on the organs, the entire skin of the abdomen was removed from the liver and the larynx. As for the back skin, 1 cm 2 of skin 2 cm from the tailing portion on the back of the skin tissue toward the head and 0.5 cm from the lumbar spine to the right was cut out, bubbling and paraffin embedded to preserve the skin tissue. Other dorsal skins were quickly placed in aluminum foil and rapidly frozen with liquid nitrogen and stored for a long time at -80 ° C.

[Test about Gene Expression Analysis]

Two dormant skins of each group were selected and total RNA samples for gene expression analysis were extracted. The dorsal skin was triturated in the presence of liquid nitrogen and dispensed into 1.5 ml Eppendorf tubes every tens of mg of wet weight. Total RNA extraction was carried out using a RNase easy mini kit (produced by QIAGEN) and followed the conventional protocol.

The extracted Total RNA was subjected to a treatment according to Affymetrix's recommended protocol, and the expression pattern in the skin tissue was examined by DNA microarray method. Specifically, cDNA is synthesized from the extracted total RNA using "SUPERSCRIPT choice system for cDNA synthesis" (trade name, manufactured by Invitrogen), and the cDNA is used as a template for "Bio Array High Yield RNA Transcript Labeling Kit". Biotin-labeled cRNA was synthesized in vitro using (trade name, manufactured by Enzo Diagnostics). After fragmenting the cRNA, hybridization is performed using a DNA microarray (trade name "Gene Chip Mouse Expression Array 430A" (manufactured by Affymetrix)) and a "hybridization oven" (trade name, manufactured by Affymetrix) to carry out R-pico. The reaction with erythrine-streptoavidine and washing were performed by a "Fluidics station" (trade name, manufactured by Affymetrix), and then the fluorescence intensity was measured by a "Gene Array Scanner" (trade name, manufactured by Affymetrix) to analyze the related gene expression amount. Was performed. In addition, the detection sensitivity of the DNA microarray is 1: 100,000, which is measured by adding and detecting a transcription product obtained from a mouse cDNA clone derived from a mouse total RNA sample. 22,690 genes were detected for the mouse skin-derived total RNA used in this test, and used for the analysis of gene expression increase and decrease. Among them, tyrosinase was encoded by Probe ID: 1448821_at, and the ratio of gene expression intensity was determined from the fluorescence intensity.

The combination of test group and control group is as follows.

Test group: Breeding for 10 weeks of MF-1 containing 0.1% Helicipron A (16 weeks of age)

Control group: MF-1 diet 10 weeks (16 weeks old)

In Table 4, "Accession No." is an identification number in GenBank (nucleic acid sequence database of NCBI) of each gene, and a probe set ID is an identification number unique to Gene Chip Expression Array manufactured by Affymetrix.

From Table 4, gene expression of tyrosinase is inhibited by helipyron A in mouse skin tissues, overexpression is inhibited in the skin of tyrosinase, and melanin formation by tyrosinase is inhibited by It can be seen that it is suppressed by.

[Measurement of Proliferation Cell Nuclear Antigen in Skin Tissue]

Proliferation Cell Nuclear Antigen (PCNA) was measured using immunohistochemical techniques.

The mouse skin tissues after paraffin embedding were deparaffinized, and the antigen-activation was carried out for 5 minutes with microwave treatment in 0.01 M citric acid buffer (pH 6.0). After cooling to room temperature, the mixture was reacted for 20 minutes in methanol containing 0.3% hydrogen peroxide at room temperature to inhibit endogenous peroxidase. After washing with water and washing with 10 mM PBS (-), blocking was performed by microwave treatment in a rabbit serum 75-fold 10 mM PBS (-) diluted solution for 5 minutes, and after the blocking was completed, the serum of the rabbit was dropped and the primary antibody (PC10: manufactured by DAKO) ) Was diluted 500-fold and the antibody was reacted by microwave treatment for 20 minutes. After washing twice with 10 mM PBS (-), the diluted biotinylated secondary antibody (Biotinylated Rabbit Immunoglobulin M; manufactured by DAKO) was added, and the antibody was reacted by microwave treatment for 5 minutes. It was washed twice with 10 mM PBS (-), and reacted by microwave treatment for 5 minutes by adding ABC reagent (ABC-HRP; manufactured by Vectastain). The reaction was carried out at room temperature for 3 minutes 30 seconds using DAB (3,3-diaminobenzidinetetrahydrochloride: manufactured by DAKO) as a color developing reagent. After washing with water, dehydration and encapsulation were performed based on a known method. The staining state of the skin tissue was observed under the microscope. In 10 weeks of ultraviolet irradiation, the nucleus of the epidermal cells developed mainly due to the increase of PCNA. Therefore, the images were read using Adobe Photoshop, and the staining points in the predetermined area of the images were quantified by NIH Imaging.

The mean value ± S.D. was obtained for the numerical values of each group. The significant difference test between the two groups was performed with the student's t-test (significance level p <0.05) after the mean value of each group was adjusted with the UV (-) group as the reference value of 1. The measured sample is shown in Table 5, and the result is shown in FIG.

There was no increase in PCNA in the skin tissue of mice fed helipyrone A. PCNA is known as a biomarker of psoriasis known as oxidation and cleavage of DNA and proliferative skin disease, and the expression of PCNA that is elevated by UV irradiation to the mouse skin in this test is fed Hhelipyron A. It returned to normal level by antioxidant activity of DNA and alleviation of ultraviolet ray disorder.

[Measurement of LoriClean in Skin Tissue]

Loriclean was measured using immunohistochemical techniques.

The mouse skin tissues after paraffin embedding were subjected to deparaffin treatment according to the conventional method, and the antigen activation was subjected to microwave treatment for 5 minutes in 0.01 M citric acid buffer (pH 6.0). After cooling to room temperature, the mixture was reacted for 20 minutes in methanol containing 0.3% hydrogen peroxide at room temperature to inhibit endogenous peroxidase. After washing with water and washing with 10 mM PBS (-), blocking was performed by microwave treatment in a 75 times 10 mM PBS (-) dilute solution of goat serum for 5 minutes, and the goat serum was dropped after the blocking was completed to remove primary antibodies (Loricrin Polyclonal Antibody, Purified: A 500-fold dilution of Sigma) was added and the antibody was reacted by microwave treatment for 20 minutes. After washing twice with 10 mM PBS (-), the diluted biotinylated secondary antibody (Biotinylated Goat Immunoglobulin M; manufactured by DAKO) was added, and the antibody was reacted by microwave treatment for 5 minutes. It was washed twice with 10 mM PBS (-), and reacted by microwave treatment for 5 minutes by adding ABC reagent (ABC-HRP; manufactured by Vectastain). The reaction was carried out at room temperature for 4 minutes 30 seconds using DAB (3,3-diaminobenzidinetetrahydrochloride: manufactured by DAKO) as a color developing reagent. Thereafter, the cell nucleus and whole tissues were stained with hematoxylin, and after washing with water, dehydration and encapsulation were performed based on a known method. The staining state of the skin tissue was observed under the microscope. In 10 weeks of ultraviolet irradiation, the nucleus of the epidermal cells develops mainly due to the increase in lyochlorine. Images were read using Adobe Photoshop, and the dyeing points in certain areas of the images were digitized by NIH Imaging. The measured sample is shown in Table 6, and the result is shown in FIG.

The mean value ± S.D. was obtained for the numerical values of each group. The significant difference test between the two groups was carried out by the student's t-test (significance level p <0.05) after the mean value of each group was adjusted with the UV (-) group as reference value 1.

Lolliclean was not increased in the skin tissue of mice fed Helipyron A. LoriClean is known as a marker of the terminal differentiation of skin, and the action of maintaining normal skin differentiation is recognized by suppressing aging of the skin by ingestion of helipyrone A.

[Measurement of keratin 1 in skin tissue]

Keratin 1 was measured using immunohistochemical techniques.

The mouse skin tissues after paraffin embedding were subjected to deparaffin treatment according to the conventional method, and the antigen activation was subjected to microwave treatment for 5 minutes in 0.01 M citric acid buffer (pH 6.0). After cooling to room temperature, the mixture was reacted for 20 minutes in methanol containing 0.3% hydrogen peroxide at room temperature to inhibit endogenous peroxidase. After washing with water and washing with 10 mM PBS (-), blocking was performed by microwave treatment in a 75 times 10 mM PBS (-) diluted solution of goat serum for 5 minutes, and the goat serum was dropped after the blocking was completed, and the primary antibody (Mouse Keratin 1 ( AF109) Polyclonal Antibody, Purified (product of COVANCE) was diluted 500 times, and the antibody was reacted by microwave treatment for 20 minutes. After washing twice with 10 mM PBS (-), the diluted biotinylated secondary antibody (Biotinylated Goat Immunoglobulin M; manufactured by DAKO) was added, and the antibody was reacted by microwave treatment for 5 minutes. It was washed twice with 10 mM PBS (-), and reacted by microwave treatment for 5 minutes by adding ABC reagent (ABC-HRP; manufactured by Vectastain). The reaction was carried out at room temperature for 3 minutes 30 seconds using DAB (3,3-diaminobenzidinetetrahydrochloride: manufactured by DAKO) as a color developing reagent. Thereafter, the cell nucleus and whole tissues were stained with hematoxylin, and after washing with water, dehydration and encapsulation were performed based on a known method. The staining state of the skin tissue was observed under the microscope. In 10 weeks of ultraviolet irradiation, keratin 1 is increased, and cell nuclei are mainly developed in epidermal cells. Images were read using Adobe Photoshop, and the dyeing points in certain areas of the images were digitized by NIH Imaging. The measured sample is shown in Table 7, and the result is shown in FIG.

The mean value ± S.D. was obtained for the numerical values of each group. The significant difference test between the two groups was carried out by the student's t-test (significance level p <0.05) after the mean value of each group was adjusted with the UV (-) group as reference value 1.

There was no increase in keratin 1 in the skin tissue of mice fed helipyrone A. Keratin 1 is known as a marker of the terminal differentiation of the skin, the action of maintaining the normal state of differentiation of the skin is suppressed by heliphyron A ingestion is suppressed.

A prescription example is shown below.

[Prescription 1 capsule]

The following components were mixed and filled in a capsule base mixed with gelatin and glycerin to obtain a soft capsule.

[Prescription 2 tablets]

The following components were mixed and tableted to obtain tablets.

[Prescription 3 tablets]

The following components were mixed and compressed to obtain a tablet.

[Prescription 4 juice]

[Prescription 5 Cream]

The following components (1) to (10) are dissolved by heating at 80 ° C to obtain an oil phase. The components (11) to (13) are dissolved by heating at 70 ° C to obtain an aqueous phase. The aqueous phase was gradually added to the oil phase to emulsify, cooled to 40 ° C while stirring, and further cooled to 30 ° C to obtain a cream.

Claims (6)

A cosmetic composition for improving wrinkles containing Helicyrone A represented by the formula (1). [Formula 1]

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