TW200810751A - 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 - Google Patents

Abstract

A new action of Helipyrone A is now found. As a useful application of Helipyrone A, the following agent is disclosed: a singlet oxygen scavenger, an anti-skin-aging agent, an anti-wrinkle agent, an anti-sagging agent, an agent for improving the moisture content in the skin, a skin-whitening agent, a melanin formation inhibitor, a nitrogen monooxide scavenger or an antioxidant, each comprising Helipyrone A.

Description

200810751 IX. Description of the invention: [Technical field to which the invention pertains] The present invention relates to a dipyridone compound A (Helipyrone A). [Prior Art] Non-Patent Document 1 (Journal 〇f ethnopharmacology 33 (1991) p51-55) discloses that the bispyranone compound A (Helipyrone A) represented by the general formula has an antibacterial action. [Non-Patent Document 1] Journal of ethnopharmac〇1〇gy 33 (1991) p51-55 [Non-Patent Document 2] Esahak Ali, et al., Phyt〇 chemistry, 1982, 21, 243-244 [Summary of the Invention] Yes, issue a new role for the double-blend A (five) calories A) and seek its effective use. The main constitution of the present invention is as follows. r mail η kinds of early feed, Xiao remover, skin aging improve H 岐 good agent, skin _ improver, skin moisture improver, whitening agent, melanin inhibitor, one oxygen = _ 槪 cap, coffee - slave (1) And the side compound A (HeliPyrone Α). 5 200810751 (Chemical Formula 2)

• A variety of oral test substances, including any of the external skin composition described in the sputum, including #员培训. (3) The food for use in any of the items described in the first aspect, which comprises the agent according to any one of the items. The pharmaceutical preparation contains any of the agents described in 1. - A cosmetic containing any of the ingredients described in i. Bi-pyrene complex A (Heli lion ne A), according to f kg confirmed that it has the elimination of singlet oxygen, improve skin aging, change her text: skin loose, improve skin water green, whitening, inhibit melanin, eliminate . The skin is protected by positive oxidation, and the coffee is used for living limbs, medicinal materials, food, and cosmetics. A new variety of uses [Embodiment] It is also represented by the following side of the compound A (Helipyrone A). , eight C1) (Chemical Formula 3) 6 200810751 General Formula (1) We can find that the double acetophenone compound A (HelipyrQneA) has anti-stranding effect, improves skin aging and photoaging, whitening agent, inhibits melanin production, T The new role of eliminating, eliminating singlet oxygen and eliminating nitrogen oxides. Based on these new ^ ^, it is used in cosmetic compositions, food additives and medical materials. As a cosmetic and food, it can improve skin aging and photoaging, whitening agents, inhibit melanin production, anti-oxidation, eliminate active oxygen, etc., and can be used as skin aging agent, silk smear, whitening agent, melanin. An inhibitor, an antioxidant y, a single oxygen scavenger, and a nitric oxide eliminator are produced. A cosmetic (including a semi-drug) containing the bispyranone A of the present invention, and if used on the face or the hands, feet, and body, the left makeup πσ can be cited as a makeup. Water, lotion, cream, gel, multi-layer makeup mouth, etc. Also, it can be used for cosmetics such as lipsticks, foundations, facial cosmetics, facial cleansers, hand washings, bathing fresh detergents, shampoos, thieves, nursing, and raw hair. In addition to the Helipyrone A, the composition of the composition of the general-purpose cosmetics is used in addition to the compound A (HeHpyr〇neA) of the present invention. . For example, it can be blended with oils such as hydrocarbon oil, cool oil, fatty acid, higher alcohol, silicone, sterol (St(10)1), ceramide, etc., polyol, sugar, and soy sauce (Pym) )lld〇necarboxylicAcid), amino acid (amin〇aci^ beet test (betaine) and other fishery agents, nonionic surfactants, anionic surfactants, cationic surfactants, amphoteric surfactants, semi-polar surfactants, (4) Grease (lecithin) surfactant, polymer emulsifier, water-soluble polymer, palmitic acid dextrin (Palraitic aciddextrin) and other tackifiers, inorganic powders, organic powders, antibacterial agents, fungicides, p Η adjusters , a sounding agent (_coffee), a coloring agent, a fragrance, a plant extract, etc. A food containing the bis-indolyl g of the present invention and a compound A (Helipyr〇ne A), which can be directly used as a compound A (Heliqing ne A), or add a variety of other nutrients as food, or (4) the desired food. For example, add the temple, lactose, maltose, vegetable oil powder, cocoa butter, stearic acid (gamma (five) Acid) After the appropriate amount of the agent, the method for the time is made into a form suitable for eating, for example, it can be formed into a granule, a granule, a suspect, a capsule, a paste, etc. as a health food, a health-care food, etc. In addition, it can also be added to various foods. Used in foods such as ham, sausage, etc. (4) foods such as jade food, fish paste, bamboo wheels, etc., processed bread, biscuits, cream, powdered milk, fermented dairy products, or added to water, juice, milk, iced drinks, etc. It is also possible to use it later. The medicine 8 200810751 containing the bi-pyrene-based compound A (fjelipyrone A) of the present invention can be combined with a solid or liquid suitable for oral, intradermal, intrarectal or injection methods. _ non-toxic carrier mixture, the second form of the investment. I beam coating agent can be listed as (4), granules, powder, capsules and other solid forms, solutions, _ agents, emulsions, etc., dry Preparations, etc., can be prepared according to the usual methods on the preparation. ~ /, the non-toxic carrier for medicine can be exemplified by glucose (f) lactose, noodles, women, mannitol (_itQl), dextrin, fat Glycerin (glycmde), polyethylene glycol (p〇lyethylene _〇1), ethyl hydroxy powder (Hydroxyethyl starch), ethylene glycol, polyoxyethylene sorbitan fatty acid ester polyoxyethylene Sorbitan Fatty Naru , amino acid, gelatin (albumen), water, physiological saline, and the like. Further, a usual additive such as a stabilizer, a 4 paste, an emulsifier, a binder, an isotonic agent, or the like may be appropriately added as needed. [Chemical synthesis of the compound A (Helipyrone A)] [Chemical synthesis of the bismuth fluorene compound A (Helipyr〇ne A), in Non-Patent Document 2 (Esahak Ali, etal·,

It has been disclosed in Phytochemistry, 1982, 21, 243-244). For example, the invention is synthesized as described below. Using 400 mL of hexane as a solvent in the presence of tetrachlorinated 9 200810751 with compound 1 · diethyl ketone (3-pentanone; 3-pentanone) 172 g, and compound 2: four Hydrogen-1, 4-oxo (tetrahydro-l, 4-〇xazine (morphine); lOOOg was reacted at 4 ° C for three hours, and purified by steaming to obtain compound 3: (N- Isopropenyl)-(N-isopropenyl- terahydr〇-l,4-〇xazine) 503.57 g, recovery 81.1%. Compound 3: (N-isopropenyl)-terahydro-l, 4-oxazine 167g and compound using 200mL of toluene as solvent 4: ethyl malonyl chloride 81 g was reacted at ice temperature (17 ° C - 11 ° C) under methanol to synthesize the compound 5 represented by the general formula 2. Chemical formula (4)

OH person General formula (2) The reaction of the compound 3 with the compound 4 is carried out by adding 2N hydrochloric acid, and after extraction with chloroform, it is subjected to a magnesium sulphate drying treatment. Next, 2 〇〇 mL of hydrazine and 25% of citric acid were sequentially added to the reaction solution, and the benzene layer was recovered by liquid-liquid partitioning. The phthalic acid layer was washed with 4 〇〇 mL of 〇1N hydrochloric acid, dried with magnesium sulfate, and then deuterated with a vacuum dehydrator (evap〇rat〇r) to obtain an orange oily solution of 200810751. One kilogram of polyphosphoric acid was added to the solution, and compound 5 was synthesized after cyclization at 110 c to 118 °C. The compound 5 was cooled to room temperature, subjected to chloroform extraction, and dried over magnesium sulfate, and then evaporated to remove chloroform. The solid was subjected to silica gel chromatography (chloroform: decyl alcohol = 50:1 (v/v)) to obtain an orange solid compound 5 of 45·7 g. 3%。 The recovery of the reaction was 18.3%. This compound 5 was polymerized by formaldehyde in the presence of 1 N hydrochloric acid. Chemical synthesis of compound 6 bispyranone compound a (Helipyrone A). The compound 5 45·7 g was dissolved in 460 mL of ethanol, and furfural (37%) 247·81 g was refluxed at 79 ° C in the presence of concentrated hydrochloric acid in a concentration of 2.5 mL to precipitate crystals. The crystals were washed with 3 mL of ethanol to give white crystals of the product of Helipyrone A (34). The yield of this reaction is η β %. The obtained bisacedanone compound A (Helipyrone®) has the properties as shown below. Appearance: white crystal NMR spectrum (400 MHz, solvent; CDC13) 卞- earning = a 1 · 231, b 1 · 969, c 2. 554, d 3. 616, e 11· 199 13C NMR (400 MHz, solvent) ;CDCh) 占=1—169.335 (t) H-168.499 (m), G->160.160 (m) 11 200810751 F-108.709 (m)5 Ε->1〇1· 592 (t) , D- >24. 368 (qt), C->19.176 (t), B-11.689 (qt), A-9.476 (q) (Formula 5)

(Chemical Formula 6)

Molecular weight: 320.341g/mol (mass analysis) Molecular formula: Cl7H20〇6 (mass analysis)

Melting point: 218 - 220 ° C [Evaluation of antioxidant energy] The evaluation of the elimination of siglet state molecular oxygen by the compound A (Helipyrone A) was carried out. The evaluation method will be 2, 2, 6, 6_

Tetramethyl-4-piperidinol (2, 2, 6, 6-tetramethy; L-4-piperidinol) (4-0HTEMP) was determined as a spin trapping agent (SpinTrapping Reagents) by spin trapping. First, the bis-compound A prepared by any concentration on a commercially available flat bottom % microplate (miCr〇Plate)

12 200810751 (Dimettiylformamide) aqueous solution 0 · lmL, followed by distilled water 〇 〇 2mL, mixed with 100 mM 4-OH TEMP aqueous solution 0. 04mL, 0 · 1mM rose red agar (rose bengal) aqueous solution 〇 · 〇 4mL, become 〇 · 2mL of the mixture. This was used as a light source for a photosensitization reaction, and a single-state oxygen was generated by irradiating a light-emitting diode (green LED) having a maximum wavelength of 530 nm for 20 minutes from the back surface of the microdisk. This mixture was transferred to a flat aqueous solution tank for ESR, and measured by an e SR spectrum. The elimination rate of singlet oxygen was determined from the intensity of the ESR spectrum, and the elimination rate was plotted against the concentration standard table to determine the enthalpy of 50% inhibition concentration I C50 . Furthermore, the existing polyphenol compound (silybin, 3, 4-dihydroxyphenylethanol, (-) catechin, (a) is simultaneously determined. ) Epicatechin gal late, quercetin, rosmarinic acid, carnosic acid, caffeic acid, tannic acid (K〇jicacid), /9-husin) IC5〇 is used as a comparison. See Table 1 for the results. In order to be clear from this result, compared with other polyantibiotic compounds, it can be concluded that the bismuth compound A (Helipqing eA) can eliminate singlet oxygen at a lower concentration. That is, it can be clearly understood that the bis-oxanone ketone compound A (Secret (4) A) has excellent singlet oxygen elimination. 13 200810751 Table 1 Sample singlet oxygen 50% inhibitory concentration (#M) Bis 11 ketone compound A 51.52 Silibinin 195. 51 3, 4-dihydroxyphenylethanol 184.86 (a) catechin 184.89 (a) a catechin catechin 111.40 quercetin 119.67 rosmarinic acid 107.06 carnosic acid 131.95 caffeic acid 278. 15 citric acid 83.15 /5 - capsin 436. 40 [melanogenesis inhibition test] Using the commercially available 6-well test JUL (well plate), mouse B16 melanoma (melan〇ma) cells were used, respectively, at 5% C 〇 2, 37 ° C, with culture medium 10% FBS (cattle). DMEM of fetal serum) was cultured to cover the entire test dish. lOOnMaMSH was added as a melanin-producing stimulating agent, and 1% dipyridone compound A was added thereto, and culture was continued for 72 hours. The solvent conditions were DMEM medium containing 1% fbs, but considering the solubility of the bis-tagpanone compound a, 0.2% acetonitrile was added in the experiment of all 14 200810751. After the end of the culture, the cytosolic fraction was recovered, and the melanin pigment was dissolved by treatment with 0. 1N sodium hydroxide, and the absorbance at 405 nm (ABS) was measured. The melanin production inhibition rate is calculated by the following formula: Melanogenesis inhibition rate [%] = 1〇〇χ{1—(ABS[Sample]) / (ABS[Control]) }

Control : MEM medium containing 〇·2% acetonitrile

Sample : Containing 〇·2% acetonitrile, adding 〇· 0001% bispyridinium-Compound A (DMEM medium) As a result, when 〇· 0001% bispyranone compound Λ was added, 221 %±&4 was inhibited. (SD·) melanin production. · [Inhibition of wrinkles caused by ultraviolet radiation] The hairless rats are continuously exposed to weak ultraviolet rays for a long period of time, and wrinkles, slacks, and the like are formed as an animal model, and tests are carried out on secrets. The compound A's feed is arrogant and its effects are resolved. L experimental animals species • system • gender species • old gas, system: H〇s: HR1 (no hair), gender: female • breeding start age 15 200810751

I

I started breeding at 5 weeks of age and started mixing feed silver food. II· breeding environment setting temperature and humidity: 24±1°C, relative humidity 55±5% Air conditioning equipment: all fresh (all fresh) lighting time: 12 hours hour automatic lighting, light out mode breeding equipment: plastic cage 5 / One cage feed: Free ingestion of powdered sterile feed for "l (oriental yeast industry (mixed or 疋MF-1 mixed with double π ketone ketone compound A (Helipyrone A) 〇. 〇 1 % (w/w) 〇· 05% (w/w), 〇·1% (w/w), or a mixture of carotenoids with excellent antioxidant effects of 0.01% (w/w), 0.05% (w/w). The test schedule ultraviolet irradiation conditions··Using an ultraviolet lamp on the next day (every day) in ten weeks, the UV-A wave is irradiated 14J/cm2 without irradiation (the irradiation time is about 1 minute), and the UV B wave is 20 mJ/cm. (Ignition time is about 50 minutes). The total exposure amount of 1 week (35 times) is UV-A wave 490J/cm2, UV-B wave 700mJ/cm2. For the group of continuous ultraviolet irradiation, it is implemented 24 hours before dissection. The last time, 'external line knowledge, shot. Anatomy 18 hours before hunger strike. Before the dissection, using a simple moisture meter (Moisture Checker) TM, scaia) to measure the amount of skin water. 16 200810751

I (with nembutal), take the appearance and take a replica of the back of the skin. After dissection, remove the liver sputum, recover the blood from the blood of the lord, and recover the back and abdomen skin. The organ is stored in a cold bundle, and the whole blood is centrifuged (3, delete g, test, room temperature), and the pl is allowed to be recovered (blood pack) for cold preservation. As for the skin, the back area is abalone. (Bouin) is the same as the other, in the rU / night mouth, for tissue dyeing, others are frozen. Focus on the appearance of the skin in the neck of the sagging (3) position (four) and back of the sensitive pattern (Wrmkle) , as defined by Bi_ DL, etc. (Ph〇t〇chem

Photobiol. (1987) Vol.46, No.3, pp367-pp78)>W^〇 In other words, '(10) touches the definition of New Zealand. The sample with the _ lying is given the intermediate value of Q. 5. The data of the wrinkle score is graphically shown in Figure 1. • 0 points Unable to recognize the occurrence of crepe. Under #止状恶, the vertical line relative to the direction of the spine cannot be recognized, or the degree of the touch can only be touched, and the secret of the side disappears. • 1 point This is enough to clearly identify the slight sensitivity. The month b is enough to distinguish the vertical line from the direction of the spine, and the line disappears as the individual moves. • 2 points 17 200810751 Clearly recognizes moderate levels of wrinkles. The moon is enough to distinguish the lines relative to the direction of the spine, even if the individual moves, the lines will not disappear and exist forever. • 3 points Clearly recognize deep wrinkles. It is enough to recognize the line relative to the direction of the spine, and can recognize the shadow of the line. Even with the movement of the individual, the line will not be; In the score of the enemy pattern, compared with the exposure group that received ultraviolet radiation for 1 week (control group), such as group is 2.4, receiving ultraviolet radiation for 1G week, and mixing with MF-1 of Q·cong and 吼 ^^ The edible group was 〇·1, and the MF-丨 edible group that received UV irradiation for 1 week and mixed with 〇·05% bis-anionone compound A was ◦·2, and received ultraviolet light: 10 weeks, mixed with Q. · The edible group of Q1% bis-anionone compound A was inhibited by the age of ''old''. 1Q Le does not irradiate the ultraviolet ray to eat MF-1 (control group) group, the enemy pattern score is 〇 · 2, through the combination of Ζ 〇 · 05~0.1 double and fine compound A, m irradiated wrinkles on the ultraviolet surface It is completely suppressed. "The silk from the skin can be transferred to know that the compound A has the effect of inhibiting the openness. The double-indolone compound A of the rat is eaten, and the ingestion of the day is used as the surface area conversion formula. y= (3ντχ) 2 is equivalent to 520mg/day/60kg body weight when converted to the amount of human administration. If 〇1% mixed feed is freely ingested, it is equivalent to about 52%/曰/60kg if heavy, yes It can be used as a food. It will not be able to take the amount of 18 200810751. In addition, the group of carrots with anti-oxidation effect does not show the effect of wrinkle suppression. The ultraviolet radiation for 10 weeks, with G· Q5% $ 胡萝卜 胡萝卜 ΜΙΜ , , , , , , , , — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — Inhibition of skin drying by ultraviolet irradiation] The skin moisture content (Moisture CheckerTM, manufactured by Scala) was measured by a simple moisture meter by a non-invasive method, and each measurement was performed five times, and the average measurement of each group was compared. The measurement results of the moisture content are shown in Fig. 2. After external exposure for 10 weeks + MF-1 (control group), the skin moisture content decreased in the sputum dry state (skin moisture content 31.4%). In contrast, UV irradiation for 10 weeks + with dipyridone compound A In the MF-1 edible group, the skin moisture content of the 0.1% dipyridone compound A was 41.3%, and the skin moisture content of the 〇·05% was 40.2%, and the skin moisture content of 0.01% was 38.2%, maintaining a high skin moisture content of about 40%. The amount of skin moisture in the edible group combined with the ketone compound A was compared with that of the MF-1 (control group) after 10 weeks without irradiating ultraviolet rays. The skin moisture content is 35·5%, and the value is higher. [Suppression of skin hardening accompanied by ultraviolet irradiation] 19 200810751 A spring type hardness tester (JIS C type) skin viscoelasticity measuring device (Vesmeter: E-100S/WaveCyber) is used. From the back part of the tail to the head 2cm, the right side of the lumbar vertebrae 5cm is measured three times to determine the average hardness. The skin hardness measurement results are shown in Figure 3. In general, hardness as an indicator of skin viscoelasticity, the number The higher the 値, the skin aging During the treatment, the skin elasticity decreased. Ultraviolet irradiation for 10 weeks + MF-1 (control group) in the edible group, the degree of skin hardening was significant (degrees 15.4). In contrast, ultraviolet irradiation for 1 week + with dipyridone The MF-1 edible group of Compound A is combined with 〇. 1% bispyranone compound a has a hardness of 8.1, and the hardness of 0. 05% is 9.1, and 〇· 〇!% The hardness degree was 9.1, and the hardness degree of the MF-丨 (control group) edible group which passed 10 weeks without irradiating ultraviolet rays was 7.2, and the degree of hardening was only slightly hardened, and the hardening was suppressed. [With 卩 | 、, 外 外 A?, inhibition of protein formation in the skin tissue (Carb〇nylafi〇n)] The carbonylated protein of one of the oxidized proteins is 〇xybl〇tTM (CHEMIC0N) To evaluate. The specific method is as follows. Weigh the mouse skin with a wet weight, and add a prcrtease inhibitor cocktail (Lisys buffer) (ρΗ=7·4) 〇·2πα at 4 °C to homogenize it. After centrifugation at 12, 〇〇〇gx2 for 5 minutes, the supernatant was filtered, and the egg self-determination was measured by the method of Bradford. Each protein 20 200810751 The sample was taken at 20/g, and its carbonyl group was pH-formed. The protein was separated by electrophoresis analysis (SDS_pAGE) and transferred to a PVDF membrane using a protein transfer device. The transferred film was blocked at room temperature for 30 minutes in a PBS (-) solution containing 5% skim milk at 4 ° C, and skimmed milk (Skimmiik) was washed with PBS (-). After the reaction, the reaction was carried out with an anti-DNPH antibody at 4 ° C overnight, and after washing, it was reacted with vitamin H (biotin) anti-mouse IgG for 1 hour. After washing, the fluorescent detection device (ECL PLUS) is used to make the film sensitive, and the image is transferred to the medical automatic image t. The result is shown in Figure 4. Ultraviolet irradiation for 10 weeks + MF-1 (control group) food test, the relative value of the county protein amount was 259.4. The 姊 , , 紫外线 紫外线 紫外线 紫外线 紫外线 紫外线 紫外线 紫外线 紫外线 紫外线 紫外线 紫外线 紫外线 紫外线 , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , The protein quality _ the value is 35.6. The complex value of the amount of 〇1% of the proteinized protein f is 1/1G~1/5. The relative value y of the amount of the egg (four) of the MF-1 (control group) consumed by the MF-1 (control group) after 1G week without irradiation with ultraviolet rays, and the relative value of the egg (four) of the flank of the compound A of the bismuth compound 'Maype's _ suppresses the formation of carbonylated proteins produced by the appearance of i-line makeup. #1=Antioxidant is used as the _ 素素, which can be known to have an inhibitory effect on the production of tick-based proteins. t Irradiation of ultraviolet rays Η) and with G Guan P - Carotenoids (4) Edible 21 200810751 'Irrigation The relative enthalpy of the amount of carbonylated protein was 153. i, 〇· ο 1 % cold-carotene MF-1 edible group, 羯190·7. [Inhibition of guanosine (8-OH dG) production by 8-hydraulic (hydrQxy) _2' in the skin tissue irradiated with ultraviolet light] 8-OHdG was measured by immunohistochemistry. Cut the tail joint of the back of the skin tissue to the head 2cm' of the right side of the lumbar vertebrae. The skin w of the 5th part of the lumbar vertebrae is fixed with the abalone liquid and embedded in the stone to preserve the skin tissue. Make a 3/zm thick slice appropriately. 'Desaturated soil and hydrophilization based on a conventional method. Microwave treatment for five minutes in citric acid buffer (pH 6.0) for antigen activation. After cooling to room temperature, at room temperature Decontamination with decyl alcohol containing 3% 3% hydrogen peroxide should be carried out for 20 minutes to block The endogenous peroxidase was washed with water, 10 mM PBS (-), and then subjected to microwave treatment for 5 minutes in a diluted solution of goat serum 75 times 10 mM PBS (-), and then the serum was removed. The antibody was reacted by microwave treatment for 1 minute with 5 times of the antibody (Μ5·1, manufactured by SEIL Co., Ltd.), washed twice with 10 mM PBS (-), and diluted with 300 times of vitamin oxime ( Biotinylation of secondary antibody (vitamin deuterated goat immunoglobulin (globulin) Μ; manufactured by DAK0), subjected to microwave treatment for five minutes to react the antibody. Wash twice with 10 mM PBS (-), add ABC test (ABC- HRP; Vectastain) is subjected to microwave treatment for five minutes to react. 22 200810751 * Use DAB (3, 3-diaminobenzidine hydrochloride: j) AK) as a color forming reagent, place three-thirds at room temperature After 10 seconds, the reaction was carried out. After washing with water, it was dehydrated and embedded in a conventional method. The staining of the skin tissue was observed under a microscope. Images were taken using M〇be Photoshop, and the staining in a certain area of the image was Nih Imaging numerical The results are shown in Fig. 5. Ultraviolet irradiation 1 week + MF-1 (control group) edible group, 8-〇H邠's phase value is 5.89. In contrast, ultraviolet irradiation 1 week + double ^ 比 的 嗣 的 的 的 的 的 的 的 MF MF MF 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 The relative value of A is the same as the value of A. The relative value of (4) is the same as the value of (.1G, 8, and the amount of dG is generated by i5/(10)~19/old age. : dG _value i•(8), which can be irradiated with ultraviolet rays, but can be combined with (8) raw 8::: shirts, and it is possible to suppress the production of ultraviolet rays by the exposure of the ground. In addition, the food has an antioxidant effect, and the formation of sediment has an inhibitory effect. ..."The group of the prime, did not show 8.::: wide _ with 〇. 〇 5% dill "sugar.i food (four) Qiu Li's relative 値 is 5 2 艮,, and the carrots (4) Food _, 8 = $1G circumference and with G. Gl%yS - "With group 8, the relative enthalpy of sputum is 23 200810751 [Suppression of lipid peroxide formation in skin tissue with UV irradiation] Fluorescence analysis TBARS (thiobarbituric acid reactive substances, nmol/g wet weight torn weight) of skin tissue was measured.枰 枰 各 各 各 各 各 各 各 各 各 各 各 各 各 各 各 各 各 各 各 各 各 各 各 各 各 各 各 各 各 各 各 各 各 各 各 各 各 各 各 各 各 各 各 各 各 各 各 各 各 各 各 各 各 各 各 各 各 各 各 各Centrifugation of lOmin. Elimination of impurities in the supernatant 'The TBA reagent (thiobarbituric acid/acetic acid buffer) was added and the reaction was heated in a boiling water bath for 1 hour. For the quantification of TBARS, a TBA reaction was carried out by using a sample containing m3-tetraethoxypropane (1,1,3,3-Tetraethoxypropane) to prepare a calibration curve. The result is shown in Figure 6. After the end of the heating reaction, it was cooled to room temperature with ice-cold, stirred with 5 mi of n-butyl alcohol, and then centrifuged at 3, OOO rpm for 10 min to recover the upper n-butanol layer. Fluorescence analysis (Ex 515 nm, Em 553 nm). The fluorescent intensity meter uses HITACHI f-2. As a result, the mass of peroxidized lipid in the tissue of the edible group was 50.8 nmol/mg in the ultraviolet irradiation for 10 weeks + MF-1 (control group), and the ultraviolet ray was irradiated for 1 week and the MF of the bis- σ-pyrone compound A was matched. 1 edible group, with 〇 · 1% bis-α 比 顚 顚 顚 Α Α Α Α Α Α Α Α Α Α 组织 组织 组织 组织 组织 组织 组织 组织 组织 组织 组织 组织 组织 组织 组织 组织 组织 组织 组织 组织 组织 同 同 同 同 同 同 同 同The mass of the peroxylipid in the tissue is 33·7 nmol/mg, and the mass of the peroxylipid in the tissue of the 0.01% bis-indolone compound A is 24 200810751 44. 8 round i/mg, peroxidation Lipid production is inhibited by 6/ι〇~9/ι〇. Ultraviolet irradiation for 10 weeks + with 〇. The consumption group of double 吼 吼 compound a and the amount of peroxide peroxide f in the 5% edible group, and MF-i (control) ribs after weeks without irradiation of ultraviolet rays The quality of the peroxidized fat in the _ is the same degree. Therefore, the dimercapto-compound A can remarkably suppress the formation of peroxidized lipids accompanying ultraviolet irradiation. In addition, gamma has an antioxidant effect as (4) 6-carotene, which has almost no inhibitory effect on the production of lipid peroxide. The MF-i edible group irradiated with ultraviolet rays for 10 weeks and with 〇·G5% A- s s s-peroxide has a mass of 46·9 nmol/mg, and irradiated with ultraviolet rays for 1 week and cooperated with MF-1 of 〇·〇1 carotene. Lnm〇1/呢。 The edible group, the quality of the lipid peroxide is 51. lnm〇1/? [Evaluation of Nitric Oxide Elimination Energy by ESR Method] For 100//M SNAP (S-Nitroso-nitro-acetyi-D, L- Penicillamine, Tongren Chemically, the nitric oxide elimination energy of the dipyridone compound A and the known (-) catechin (manufactured by Sigma) as an oxidation preventing agent were compared. Regarding the spin trapping agent, a mixed N-methyl-D-reduced glucamine-dithiocarbamate (hereinafter referred to as MGD. purchased from LAB0TEC (share)) and iron sulfate (using (MGD) 2-Fe2+ complex prepared by π) aqueous solution. This complex is formed by reaction with nitric oxide (MGD) 25 200810751

The complex of I-gluco-2-Fe2+-NO produces an ESR spectral intensity, so the elimination energy of nitric oxide is measured by measuring the ESR spectrum of the respective concentrations of the nitrogen oxide eliminator. 50mM iron sulphate monohydrate (iron) (iron (sulfur) sulfate n-hydrate) solvent · steaming water 100 / / L (last concentration of 5mM), 50mMMGD solvent PBS (ρH7 · 4) 100 / L (after the 〉 chen Degree 5 mM), test substance solvent: 10% acetonitrile (acet〇nitri le) 100 / / L, 100 mM phosphate buffer 699 / / L, 100 mM SNAP solvent 〇 · 1M sodium hydroxide 1 / L (dare concentration i 〇〇#μ) A microcentrifuge tube (Eppendorf Tubes) of 1.5 ml capacity was sequentially added, and the reaction was stirred at 25 ° C for 10 minutes from the addition of SNAP. Nitrogen oxide was evolved from SNAP due to a 10 minute 25 ° C reaction, and the signal intensity ratio of (MGD) 2-Fe2+-NO was composed of three peaks of =i:1:1. This signal intensity is based on how much the test substance is increased or decreased to evaluate the nitric oxide elimination energy. Specifically, based on the signal intensity (C) of the control group to which the test substance is not added, the signal intensity (S) at the time of adding the test substance is determined according to the following formula, as shown in FIG. 0 Nitric oxide elimination rate = (1 - S / C) xlOO (%) As a result, the ratio of di-pyrene-pyrene to the same compound A in the range of 25//Μ~200//Μ, the concentration-dependent nitric oxide elimination rate Increase (in 25//Μ is 32.4%, 50/Μ is 52.6 %, 100//Μ is 63.6%, and 2〇〇4 is 80.1%), IC50 concentration is 51· 4 //Μ 'relative to this' catechins, 100//Μ, the elimination rate of nitric oxide is only 5.6%, 11·, respectively, mo#μ is 31.4%, and 500 is 26 200810751

I

I 47· 4% nitric oxide elimination rate. The IC50 concentration was calculated to be 547//M. The machine and machine condition settings used this time are as follows. Machine: JEOL JES TE200, X-band ESR device (ΙΟΟΚΗζ, manufactured by JEOL Ltd.) Machine condition setting: Center field: 335. 0±5. OmT, microwave power: 4mW, modulation amplitude modulation amplitude: 0· lmT, gain gain: 500, time constant time constant: 0·1 second, scanning time: scanning time: 1 minute [Nitric oxide elimination energy using colorimetry (Griess method)] Nitric oxide present in the bath solution in aqueous solution It is extremely unstable, so in addition to the ESR method, the amount of nitric oxide is indirectly evaluated by measuring the nitrite ion as an oxidizing oxide. At the same time, (-) catechin, maltol, and c〇umaric acid, which are known as antioxidant substances, were evaluated, and compared with the di-indole A. According to the measurement of the Griess method, the Assay Kit - Cn (Colorimetric) (manufactured by Toray Chemical Co., Ltd.) was used. Use N0C-5 (1-hydroxy(hydr〇xy)_2_oxy(〇χ〇)_3-aminopropyl(-__yl))-3-isopropyl (iSQPn)pylM_triazide (Wne)) 27 200810751 j, manufactured by Tongren Chemical Co., Ltd.), as a nitrogen oxide generator, until the use of a bath solution of ruthenium oxide is used at a temperature of 2 Torr. Cold; East save. The nitrogen oxide is produced by diluting (10) (10) times the buffer in the reagent group (referred to as "buffer" below pH-7·6') to the _ domain. N0C-5, which was developed as a nitric oxide donor (4), is stable in the test solution, but under neutral and acidic conditions, it is an unstable compound that generates and oxidizes (25 degrees, at ρΗ7· 4 conditions under half-life). The test substance solvent was added to a commercially available flat-bottom 96-well culture dish. The buffer containing 1% acetonitrile (ρΗ=7·6) 40//L was used to change the concentration of the test substance. After that, add 20/8 L buffer (ρη=7·6), add 1 〇〇# ΜN〇c-5 solution 4〇“L reacts with 2 times of strong C. After the reaction, add 5 〇 Place the test chamber A in the reagent group for 5 minutes, continue to add 50 μl of Griess reagent β, and place 1 〇" minute for color reaction (CQlor reaetiQn). The absorbance at 540 nm was measured on a multi-function microdisk analyzer. The absorbance at the added concentration of each test substance was s (10), not = the absorbance of the control group of the test substance was C54 〇, and the blank of the test substance and N〇c-5 was not used. The nitric oxide elimination rate was calculated by the following formula, as shown in Table 2 and Figure 8. - Nitrogen oxide elimination rate (%) = lGGx (l - (S54. - B54 〇 / / (Cs4 - - β54.)) The calibration curve is replaced by NOC-5 solution of sodium nitrite solution in 〇~1〇〇 The absorbance of the sample set in the concentration region of #Μ is determined to determine the concentration of nitrite ions in the solution. The concentration of nitrite ions can be used to determine the nitrogen concentration of the oxidized 28 200810751 produced by the NOC-5 solution. The intensity of the nitrogen elimination energy was compared with the concentration of 50% (IC50, / / Μ) in the experimental system. From 100 / / Μ N0C-5 solution, the formation of 40.7 / / Μ nitric oxide As the nitrite ion, at this time, according to the decrease in the absorbance of the test substance added, that is, the elimination activity of nitric oxide is shown. Table 2 Addition concentration UM) Nitric oxide elimination rate (%) Bis-indanone compound A (-) catechin maltol p-coumaric acid 400 79.4 69.7 75.8 73.4 300 73.7 64.5 70.9 64.7 200 69.4 47.1 66.3 46.2 100 64.9 27.5 51.2 33.5 50 45.4 18.3 36.3 26.9 Bipyranone compound Α at 50// Μ~400//Μ range, it can be seen that its concentration depends on the elimination of nitric oxide, IC50 thick 3//Μ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ ◦ IC IC 190 190 190 190 190 190 190 190 190 190 190 190 190 190 190 190 190 190 190 190 190 190 190 190 190 190 190 190 190 190 190 190 190 190 190 190 94. . It can be confirmed that the bispyranone compound Α has excellent nitric oxide elimination energy. 0 29 200810751 ^ > It is known that the bis-π-pyramid compound A of the present invention can clearly eliminate - emulsification II, as a vascular system, nerve Systematic, immunological agents are quite useful. The double yarn_compound A has the side that eliminates the excessively normal recovery of the oxygen domain in the living body, and the premature administration of the _-oxygen listener to prevent the production of excess nitrogen oxide in the living body. . That is, in the present specification, the oxygen-aged energy of the compound A in the double (four) side does not mean that all the nitrogen oxides in the body of the sister are completely eliminated, but that the excess nitrogen oxide in the living body is eliminated, and the scale is shouted. Maintain proper weight for the body. [Persistence of peroxynitrite elimination by colorimetry] The peroxinic nitrite elimination ability of the dipyridyl-ruthenium complex A is based on the Griess method's use of N〇2/N〇3 reagent Group (AssayKit) (Colorimetric) (manufactured by Tongren Chemical Co., Ltd.). The known antioxidant substances (_) catechin, maltol, p-coumaric acid were also evaluated, and compared with the dipyridone compound A. The test substance solvent was added to a commercially available flat-bottom 96-well culture dish: a buffer containing 丨% acetonitrile (ρΗ=7.6) 40//L to change the concentration of the test substance. Thereafter, a reductase 10//L and a coenzyme 10//L in the reagent group were added to the respective wells. Add 40//L of SIN-1 (3-(4-Merminyl Morpholinyl) Sydney imine sydnonimine hydrochloride) 50//M as peroxynitrite to produce 30 200810751 9 agent' to make it react at 37 C 2 hours. After completion of the reaction, Griess reagent A in the 50//L reagent group was added, and the mixture was allowed to stand for 5 minutes. Then, 50/L of Griess reagent B was added and left for 1 minute to carry out a color reaction. The versatility of the 54 〇 ship was measured on a multi-function microdisk analyzer. The absorbance of the X test substance is defined as 4. The absorbance of the control group containing no test substance was C540, and the absorbance of the blank group containing no test substance and SIN-1 was b540, and the peroxynitrite elimination rate (%) was obtained by the following formula, as shown in Table 3. Figure 9 shows. Peroxynitrite elimination rate (%) = 100x (1—(S540 — B540) / (C540 — B540)) The calibration curve is a concentration range of 0 to 100//M in the sodium nitrate solution instead of the SIN-1 solution. The absorbance of the set sample is determined to determine the concentration of nitrate ions in the solution. The concentration of peroxynitrite produced by the SIN-i solution can be determined from the nitrate ion concentration. The intensity of the peroxynitrite elimination energy was compared by eliminating the concentration of 5〇% (IC50, //M) in the experimental system. As a result, in the control group, peroxynitrite 8.18 //M ' from 50#M of SIN-1 was produced, and the coffee concentration of 5 〇% of its peroxynitrite was eliminated, which was 24 with respect to the dioxanone compound A. 04/zM, maltitol is 31.84 mM, p-coumaric acid is 31 sen #M, () 儿余素 is 705. 6yM' shows that the bispyranone compound a has excellent peroxynitrite Eliminate energy. 31 200810751 t Table 3

[Measurement of obstruction of guanaminease activity]; Flat-bottom 96-well culture 胍 Add test substance solution i 〇〇 "L, add 6 (9) units 32 200810751 /ml End from the 4 (Agaricus bisporus) of the tyrosinase (tyrosinase ) 10 / / L, cultured at 30 ° C for 1 minute. After that, add 6 mM (L-DO3 (L-K3, 4-diphenyl) Alanine solution 100//L, incubated at 3 (rc) at intervals of 4 、, - cultivating. The vehicle used for the test substance contains: % acetonitrile i _M sulphate acid buffer (ρΗ5· 5 The reagent for 'tyrase and L-D〇pA is succinic acid buffer (pH 5·5). After shaking, the absorbance is measured at a wavelength of 475 nm of the melanin amount. S475 stands for: No control substance added is the control group (6), its absorbance is a?5, no L-D0PA is added as the sample blank group (SB), its absorbance is SB"5, no added sample and L- The case of D〇pA is the control blank group (cb), and its absorbance is CB475, and the guanaminease inhibition rate (%) is calculated by the formula 2. Using arbutin (a) Rbutm) (Tokyo Chemical Industry Co., Ltd.) as a positive control group. Blocking rate of tyrosinase activity (%) U-...-SBW /(6) -CB475) } },,,,,,,,,,,,, The lutein of the lmM is higher than that of the ketone compound A and arbutin, and is 36.34% in the di-ketone compound A, and the arbutin is 25.77. Compound a is superior to arbutin in that it has an excellent hindrance and hindrance, and shows a whitening effect of double-labeled b. A. [Analysis of the expression of the catalinase gene in mice] Let the mice ingest the bis-indolone compound A When measured in mouse skin will be amineamine 33 200810751 « * Acidase-encoded gene expression (geneexpressi〇n). I · Laboratory animal system, first, sex: species · mouse, system: Hos · HR1 ( Hairless η mouse), gender: female ",, small = test _ starting week age: 5 weeks old (after cultivating 丨 丨 from __ outside line 1-3) Microbial level: sPF 1 4 · Feeder · Qingshui experimental materials Co., Ltd. 饲·feeding environment Hanjing temperature and humidity: 24±l°c, relative humidity 55±5% Air conditioning equipment: new wind Type of time: 12 hours of automatic lighting, xenon lamp breeding equipment: 5 plastic cages / 1 cage feed · Free intake of powdered sterilized feed MF-1 (ORIENTAL Yeast Industry Co., Ltd.), also prepared mixed 〇 · 1% by weight of the compound of the bismuth and bismuth compound. η Subject matter administration: It is allowed to freely ingest the powder mixed feed. The fasting begins from the anatomy π hours ago. Water supply: free intake of sterilized tap water 34 200810751

I IV-3. Anatomy The weight of the individual who started the fasting 18 hours before the anatomy was measured by the intraperitoneal injection of pentobarbital anesthesia (40 mg/kg): into the drunk. The cut section was heparinized from the heart for blood collection. Next, the internal organs were visually observed, and the entire back of the buttocks was taken out from the liver and the back of the head. Regarding the back skin, the root of the tail on the back side of the skin tissue is 2 cm toward the head, and the skin from the lumbar vertebrae is cut from the lumbar vertebrae to the right side. The skin of the 5 cm portion is cut into 1 cm 2 , fixed by the bow sound, and the skin tissue is preserved after the stone rail is buried. Other back skins are quickly placed in ISf|paper, and the job-reduction chills are stored in -8()<t for a long time. [Experiment on Analysis of Gene Expression] From each group, the back skin of the two tissues was selected, and Total RNA samples for gene expression analysis were extracted. It was finely chopped in the presence of liquid nitrogen, and a microcentrifuge tube (eppend〇rf plus (6) of a volume of 1.25 ml was injected at a wet weight of several tens of mg. The RNase easy mini kit (manufactured by QIAGEN) was used to follow the general procedure. Total calendar. The total RNA extracted was processed in the order suggested by Affymetrix, and the expression pattern of the skin tissue was investigated by the microarray method. Specifically, the SUPER RNARI selection was used from the extracted Total RNA. System for cDNA synthesis" (trade name, 35 200810751 t

I

Invitrogen company to synthesize cDNA, use this cDNA as a template, use "Bio

Array High Yield RNA Transcript Labeling Kit" (trade name, Enzo

Diagnostics, Inc., synthesizes cRNAs that recognize vitamin H in vitro. As a result, after fragmenting this cRNA, the DNA microarray (trade name rGeneChip)

Mouse Expression Array 430A" (manufactured by Affymetrix Co., Ltd.) and Hybridization Oven (trade name, manufactured by Affymetrix Co., Ltd.) were hybridized and cultured, and "Fluidics station" (trade name, manufactured by Affymetrix Co., Ltd.) and R-algae After the reaction and washing operation of R-phyC〇ery^rin Streptavidin, the fluorescence intensity was measured by rGene Array Scanner (trade name, manufactured by Affymetrix Co., Ltd.), and the amount of related genes was measured. Analysis. Further, the detection sensitivity of the above DNA microarray was: 100,000, which was determined by adding a copy of the mouse (9)-like replica and the identified completed copy product in the total RNA sample of the mouse. About the total RNA from mouse skin used in this experiment, 22,69 genes were detected for analysis of the increase or decrease of gene expression. The tyrosinase was encoded as pr〇beID: 144882-at' to determine the gene expression intensity from its fluorescence intensity. The combination of the experimental group and the control group was as follows: The test group: MF-丨 food containing 0·1% bispyranone compound A was fed for 1 week (16 weeks old) Control group: MF-1 food was fed for 1 week (16 weeks old) 36 200810751 > * Table 4 Probe Probe Login No. Accession No· (Human human) Login No. Accession No· (mouse mouse) Control group food intake double. Ratio 喃 _ compound A ratio Ratio (ingestion double ° 比 ketone compound A / control group food) 1448821_at NM—000372 NM—011661 1276.9 479. 75 0. 37575

In addition, "Accession No." in Table 4 is the identification number of each gene in GenBank (NCBI's Nucleic Acid Arrangement Database), and Probe Set ID is the unique identification number of the GeneChip Expression Array manufactured by Affymetrix. As can be seen from Table 4, the gene expression of tyrosinase on mouse skin tissue was inhibited by the dipyrone ketone compound A, and the excess performance of tyrosinase present on the skin was inhibited due to tyrosinase The melanin formed is inhibited by the dipyridone compound A. [Measurement of Proliferation CeH Nuciear Antigen in Skin Tissue] Proliferating cell nuclear antigen (PCNA) is chemically determined from immune tissues. Regarding the skin tissue of the mouse which had been embedded in the paraffin pack, it was subjected to dewaxing treatment according to a conventional method, and microwave activation was carried out in a 1.0 M citrate buffer (ρΗ6·〇) for five minutes to effect antigen activation. After cooling to room temperature, it was neutralized at room temperature for 20 minutes with decyl alcohol containing 〇·3% peroxygen gas to hinder its endogenous peroxidase. After washing with water and 10 mM PBS (-), the rabbit serum was 75 times 1 〇 ρ ρ 37 200810751 release / 谷液贝 for 5 minutes of microwave treatment, after the interval was separated, the rabbit serum was removed, and the dilution was added 500 times once. The antibody (manufactured by PC10··DAK0) was reacted by microwave treatment for 20 minutes. The cells were washed twice with 1Q mM pBS (-), and diluted with 3Q (H-port, ginseng-nized secondary antibody (vitamin η-treated rabbit immunoglobulin M, manufactured by DAK)), and subjected to microwave treatment for 5 minutes. The reaction was carried out by washing twice with 10 mM PBS (sj, adding hydrazine test (ABC-HRP; manufactured by VeCtastain) for 5 minutes by microwave treatment. (4) AB (3, 3-diaminobenzidine tetrahydrochloride) : DAK0) is a color forming reagent, which is allowed to stand at room temperature for 3 minutes and 30 seconds to react. After washing with water, it is dehydrated according to a conventional method, and is sealed. The staining state of the skin tissue is observed under a microscope. In the sample of 10 weeks of the week, due to the increase of PCNA, the epidermis cells were colored, and the field I was colored. The image was taken using Adobe Photoshop, and the stained area in the image and the inside of the image was quantified by NIH Imaging. The average value of each group is determined as SD. The significant difference between the two groups is determined by the uv (-) group as the reference value. After correcting the average value of each group, 3 plus is performed, s est (" , and the level of the staff ρ < 〇 · 〇 5). The sample is shown in Table 5. The results are shown in the figure. The mice ingested with the bispyranone compound 并未 did not increase the pCNA in the skin tissue. PCNA can be used to oxidize or cut the surface, and as a patient with skin disease The performance of the well-known, cognac biomarker increased. In this experiment, the PCNA of the mouse's skin was irradiated with ultraviolet rays, and the PCNA was increased due to the consumption of the dipyridamole 38 200810751. The ultraviolet radiation was alleviated as the DNA produced by the ketone compound A. Oxidation prevention function, and return to normal level. Table 5 1 - Unirradiated UV control group food 2 UV irradiation - - Control group food ^~~ 3 UV irradiation mixing 0·1% double, _ compound 4 UV irradiation Mix 0. 05% double samarium compound '^ 5 UV irradiation mixed 〇.G1% doubling <axisization 6 UV irradiation mixed 〇· 05% /3 carotene feed one ^ 7 UV irradiation mixing 0.01%/3 Feed of carotene - 'Determination of loricin in skin tissue】 The protein of the scorpion protein is chemically determined from the immune tissue. The mouse has been buried in the stone worm. The skin tissue was subjected to microwave treatment in a 0.01 M citrate buffer (pH 6. 〇) for 5 minutes to carry out the original activation. After cooling to room temperature, it was contained at room temperature to contain G. 3 . % of hydrogen peroxide is reversed for 20 minutes, which interferes with its _ (four) Weiwei enzyme. After washing, _ PBS (-) after washing, it is carried out for 5 minutes with a diluted solution of goat serum 75 times. The cells were isolated by microwave treatment, and the goat serum was removed after the separation. The antibody was diluted 500-fold (Loricrin Polyclonai Ar^body, manufactured by Sigma). The antibody was treated by microwave treatment for 2 minutes. . Thereafter, the cells were washed twice with 10 mM PBS (-), and a secondary antibody (vitaminated goat immunoglobulin oxime; manufactured by DAK0) diluted with 3 valavirin was added thereto, and subjected to microwave treatment for 5 minutes to react the antibody. The cells were washed twice with 1 mM PBS (~), and ABC test (ABC-HRP; manufactured by Vectastain) was added for microwave treatment for 5 minutes to cause a reaction. DAB (3,3-diaminobenzidine hydrochloride: manufactured by DAK) was used as a color forming reagent to allow the reaction to stand at room temperature for four minutes and thirty seconds. The nucleus or tissue is then stained with Hematoxylin. After washing with water, it is dehydrated based on a conventional method and subjected to encapsulation treatment. The staining of the skin tissue was observed under a microscope. After 10 weeks of exposure to ultraviolet light, the main nucleus of the epidermal cells is colored due to the increase of the armor protein, and the image is obtained using Ad〇be ph〇t〇sh〇p, and the staining area in a certain area of the image is obtained. , digitized by NIH Imaging. The measured samples are shown in Table 6, and the results are shown in Figure π. The values of the respective groups were averaged ± S.D. The two groups _ significant difference test was based on the claw (-) group as the reference value 1. After correcting the average value of each group, implement student, s t-test (significant level p < 〇. 〇 5). The mice ingested with pyrone compound A have no thyroin in their skin tissue. They are known as biomarkers for terminal differentiation of skin, and can inhibit the reduction of females due to ingestion of double-blends. Aging phenomenon, and maintain normal skin 200810751 Table 6 1 Unirradiated UV--- Control group food ~ 2 UV irradiation control group food c\ —~~-~~_ UV irradiation ^-------- 3 Mix 0·1% bismuth oxime compound Α feed 4 UV irradiation ----------- Mix 0· 05% bis-indolone compound a feed 5 UV irradiation-~~~-__ - - Mix 0. 01% dipyridone compound A. 6 UV irradiation ------- Mix 0.05% yS carotene feed 7 Prepare, mix externally with 0.01%/5 carotene feed [skin Determination of keratin 1 in tissues] Keratin type 1 was determined by immunohistochemistry. The skin tissue of the rats that have been buried in the stone bag is treated according to the conventional method, and the antigen is activated by microwave treatment for 5 minutes in a 〇· 01M citric acid solution (ρΗ 6· 〇). After cooling to room temperature, the decontamination at room temperature with methanol containing 3% 3% hydrogen peroxide should be 2 G minutes, and the peroxidase which is obstructive to it. After washing with water and lOmM PBS (-), the diluted solution of goat serum 75 times pBS) was subjected to microwave treatment for 5 minutes to separate it. After the separation, the goat serum was removed, and the antibody diluted 500 times was added. (Mouse Keratinl (AF109) Polyclonal Antibody, Purified · C0VANCE), reacted with antibodies for 20 minutes of microwave treatment. The cells were washed twice (-), and a secondary antibody diluted with 300 times of vitamin η (vitamin η goat immunoglobulin m; dak〇 41 manufactured by 200810751) was added to carry out microwave treatment for 5 minutes to react the antibody. Two people were washed and challenged with 1 mM PBS. The Age test (ABC-HRP; manufactured by Vect as tain) was subjected to microwave treatment for 5 minutes to cause a reaction. DAB (3,3-diaminobenzidine hydrochloride: DAK0 is used as a color forming agent, and allowed to stand at room temperature for 3 minutes and 30 seconds for reaction. After that, the nucleus or tissue is stained with hematoxylin. After washing, based on the habit The method of dehydration is carried out, and the encapsulation treatment is carried out. The staining state of the skin tissue is observed under a microscope. The sample which is detected by the I line for 1 week, the main, and the nucleus of the epidermal cell due to the increase of the keratin type 1 Coloring, using Ad〇be Photoshop to obtain images, the staining area in a certain area of the image is quantified by NIH Imaging. The measured samples are shown in Table 7, and the results are shown in Fig. 12. The numerical values of each group are obtained. Mean ± SD. The significant difference between the two groups was determined by using the uv (-) group as the reference value 1. After correcting the average of each group, implement student, st-test (significant level p < 〇〇 5). The keratin 丨 type in the skin tissue of the mice with pyryl ruthenium compound A was not increased. Xiao egg from the type 1 is known to be a marker of skin terminal differentiation and differentiation, and can inhibit the incorporation of bis and π ratios. Skin aging and keep normal skin 42 200810751 Table 7 1 Unirradiated UV - _ ' - -~~~~~ - Control group food 2 UV irradiation ~~~______ Control group food 3 UV irradiation - Mix 0·1% double and mutter _ compound Α feed 4 UV irradiation - ^ Mix 0 · 05% double whisper _ compound A feed 5 UV irradiation --- _ mixed 0 · 01% double whisper _ compound A feed 6 ---- UV Irradiation--S_______ Mixing 0.05% yoghurt feed 7 ~------UV irradiation——^___ 心合〇· 01%/5Carotene feed------- The prescription is as follows: [Prescription Example 1: Capsule] The following ingredients are mixed and filled into a capsule base in which gelatin and glycerin are mixed to obtain a soft capsule (combination amount; mg) 70 30 10 110 (composition) And "Biam_Compound A Triene tocopherol wax seed oil [Prescription Example 2 ··Latch] The following ingredients are mixed and tableted into a tablet. 43 200810751 (Composition) Bis(A) ketone compound A Cellulose powder (combination amount; mg) 75 40 20 sucrose fatty acid ester 2 [Prescription Example 3: Lozenge] Mix the following ingredients Ingots, tablets, tablets, tablets, tablets, tablets, ketones, sucrose, sucrose, sucrose, sucrose, sucrose, sucrose, sucrose Ascorbic acid citrate (L-Ascorbic acid) Perfume (combination; mass%) 5.00 10.40 0.20 0.02 Pigment 0.10 44 200810751 Double 吼 _ _ Compound A Water 0.05 84.33 [Prescription Example 5: Cream] into eight ingredients (1) ~ (1 ° ) dissolve, force gamma. C makes it an oil phase. Dissolve the knife ~ (13) and heat it to make it an aqueous phase. Slowly add to the oil phase to emulsify, - secretly cool to the boot, more into the butterfly - phase to 30 C to get the cream. Semi-order (1) stearyl alcohol (2) stearic acid (3) hydrogenated lanolin (4) squalan (5) dioctyldodecanol ( 〇ctyi d〇decan〇1) (6) POE (25) cetyl alcohol ether (7) glyceryl monostearate (8) bispyranone compound a (9) Preservatives (10) Perfume (11) 1,3 Butanediol (12) PEG 1500 (13) Refined water 6. 2· 10 0· Appropriate amount 6.0 Residue 45 200810751 [Simple diagram] Figure 1 A chart showing the sensitivity score. Fig. 2 is a graph showing the amount of skin moisture. Fig. 3 is a graph showing skin hardness. Figure 4 is a graph showing the amount of protein (relative to enthalpy). Fig. 5 is a graph showing the amount (relatively enthalpy) of the 8-trans-base 2,-deoxyguanine nucleus in the skin tissue. Figure 6 is a graph showing the quality of peroxidized lipids in skin tissue. A graph of the elimination of nitric oxide produced by the Qing method. Fig. 8 is a graph showing the nitric oxide elimination energy by the colorimetric method (Griess method). Figure 9 is a graph showing the colorimetric peroxynitrite leakage energy. Fig. 10 is a graph showing the increase in inhibition ability of PCNA. Figure 11 is a graph showing the inhibition ability of Table 7F. Fig. 12 is a graph showing the stimulating power of keratin type 1. [Main component symbol description] 46

Claims (1)

200810751 * I X. Application for Patent Park: 】.- Type single H preparation remover, skin aging change, _ change money, skin pine improver, skin moisture improver, whitening agent, melanin anti-adhesive, mono-oxidation The nitrogen-eliminating agent or the oxidation preventing agent contains the bis-indolone compound A (Helipyrone A) represented by the general formula (1). (Chemical Formula 1) 〇H 0H 0 (; (general formula (1)) 2 - a composition for oral use, containing any of the agents as described in the scope of the patent application. 3. A skin external composition, such as a patent application The agent according to any one of the items of the first aspect of the invention, wherein the food product contains the agent according to any one of the first aspect of the patent application. Any one of the agents described in the item. 6. A cosmetic comprising any of the agents described in the first item of the patent application.