Patent Application
20190038572
This application claims the priority of Korean Patent Application No. 2012-69710 filed on Jun. 28, 2012, in the Korean Intellectual Property Office, the disclosure of which is incorporated herein by reference.
The present invention relates to an ultraviolet absorption composition capable of inhibiting skin damage caused by ultraviolet and protecting skin from ultraviolet.
In general, when ultraviolet included in sunlight is excessively and directly exposed to skin, a formation of red spots or a production of melanin in skin cells may be promoted to cause a generation of freckles or blemishes spots. Further, sebum secreted in epidermis is reacted to produce lipid peroxide, and thereby skin problems maybe caused. Furthermore, in severe cases, skin cancer may be caused. Ultraviolet rays are classified into UV-A (320 nm to 400 nm), UV-B (280 nm to 320 nm) and UV-C (200 nm to 280 nm) according to a wavelength, and among them, it has been known that ultraviolet that reach the top of the ground and then affect a human body are UV-A and UV-B.
It has been known that an UVB exposure allows free radicals to be greatly produced and reactive oxygen species (ROS) to be greatly generated in skin, induces oxidative stress to cell components, such as DNA, cell membrane, and protein, and thus ages the skin. For this reason, various studies on natural antioxidants for protecting skin damage induced by UVB are currently underway.
Meanwhile, a 3-Bromo-4,5-dihydroxybenzaldehyde (BDB) can be isolated from red alga such as Rhodomela confervoides, Polysiphonia morrowii, and Polysiphonia urceolata. The BDB has an antiviral effect to hematopoietic necrosis virus and infectious pancreatic necrosis virus, and also a 1,1-diphenyl-2-picrylhydrazyl radical removal effect. However, an effect of the BDB in protecting against UVB is not known.
Patent Document 1: Korean Patent Publication No. 2002-0042020
Accordingly, the inventors of the present invention found that a BDB has an anti-oxidative effect according to free radical removal activity in a cell, in which the free radical is generated due to an irradiation of ultraviolet, an effect in absorbing such ultraviolet itself, and also a cell protective effect according to an inhibition of apoptosis induced by an irradiation of ultraviolet. Thus, the inventors completed the present invention.
Accordingly, an object of the present invention is to provide a pharmaceutical composition for inhibiting and treating skin damage caused by ultraviolet, in which the composition includes a 3-bromo-4,5-dihydroxybenzaldehyde (BDB) or salt thereof as an effective component.
In addition, another object of the present invention is to provide an ultraviolet absorption composition including a 3-bromo-4,5-dihydroxybenzaldehyde or salt thereof as an effective component.
Still another object of the present invention is to provide a cosmetic composition for inhibiting skin damage caused by ultraviolet and protecting skin from ultraviolet, in which the composition includes a 3-bromo-4,5-dihydroxybenzaldehyde (BDB) or salt thereof as an effective component.
In order to achieve the objects of the present invention as described above, according to an aspect of the present invention, there is provided a pharmaceutical composition for inhibiting and treating skin damage caused by ultraviolet, in which the composition includes a 3-bromo-4,5-dihydroxybenzaldehyde (BDB) or salt thereof as an effective component.
According to an example of the present invention, the 3-bromo-4,5-dihydroxybenzaldehyde may have activity of removing intracellular free radicals generated by an ultraviolet absorption and ultraviolet irradiation or activity of inhibiting apoptosis induced by ultraviolet in a cell.
According to an example of the present invention, the pharmaceutical composition may be a composition in a type for external application of skin selected from the group consisting of cream, gel, a patch, a spraying agent, ointment, a hardening agent, lotions, liniments, pastes, and cataplasma.
According to an example of the present invention, the composition may include a BDB in a concentration of 10 μM to 40 μM.
In addition, the present invention provides an ultraviolet absorption composition including a 3-bromo-4,5-dihydroxybenzaldehyde or salt thereof as an effective component.
Furthermore, the present invention provides a cosmetic composition for inhibiting skin damage caused by ultraviolet and protecting skin from ultraviolet, in which the composition includes a 3-bromo-4,5-dihydroxybenzaldehyde or salt thereof as an effective component.
According to an example of the present invention, the cosmetic composition may be formulated into skin lotions, skin softeners, skin toners, astringents, lotions, milky lotions, moisture lotions, nutrition lotions, massage creams, nutrition creams, moisture creams, hand creams, essences, nutrition essences, packs, soaps, shampoos, cleansing foams, cleansing lotions, cleansing creams, body lotions, body cleansers, milky liquids, lipsticks, make-up bases, foundations, press powders, loose powders, or eye shadows.
According to an example of the present invention, the composition may include a BDB in a concentration of 10 μM to 40 μM.
The above and other aspects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:
Exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings.
The present invention was completed by confirming that a 3-bromo-4,5-dihydroxybenzaldehyde (BDB) had an effect in inhibiting oxidative stress of cell by inhibiting a production of reactive oxygen species caused by ultraviolet B (UVB), so that the BDB improves cell viability of cell (human epidermal keratinocyte), ultimately inhibits apoptosis caused by ultraviolet rays, and protects cells.
It has been known that UVB induces oxidative stress by producing reactive oxygen species, and thus induces damages of various skin organization cells. For this reason, a photo-aging process is ultimately accelerated. According to the present invention, it was found that the BDB of the present invention is suitable as the composition having an antioxidant effect through comparison experiments of free radical removal ability, UVB absorption ability, ability of inhibiting cytotoxicity and oxidative death caused by UVB irradiation, and an intracellular antioxidant effect.
More specifically, according to an example of the present invention, it was confirmed the fact that the BDB of the present invention inhibits apoptosis caused by UVB, and thus increases cell viability depending on its concentrations.
UVB light accelerates ROS generation and induces oxidative stress. A treatment of BDB is effective in inhibiting oxidative stress induced by UVB radiation in skin keratinocyte. When the BDB is treated, the BDB removes ROS in the cell exposed to UVB irradiation, so that a production of ROS is reduced. According to an example of the present invention, it was confirmed that the BDB removes intracellular DPPH radicals, superoxide anions, hydroxyl radicals, and ROS.
A cell protective effect of a BDB is relevant to UV absorption ability as illustrated in an absorption spectrum of the BDB. Therefore, the BDB can decrease the number of photons that attack a cell. Among many light protectors, natural antioxidants can effectively decrease oxidative skin damage caused by UVB. Accordingly, according to the present invention, it can be confirmed that the BDB can protect skin cells by directly absorbing UVB.
A BDB is a phenol-based compound and has an antioxidant effect by removing ROS. Cell damage caused by UVB is multifaceted. For example, cell membrane lipids are prone to be damaged by UVB. A BDB protects cell membrane lipid from UVB. In addition, UVB radiation induces fragmentation of DNA strand. According to an example of the present invention, the BDB exhibits small DNA tail confirmed by a comet assay. Accordingly, it may be inferred that the BDB of the present invention inhibits lipid peroxidation to protect a cell from UVB.
Protein carbonylation functions as a biomarker in the protein damage induced by oxidative stress. Further, when modified protein carbonyl groups are accumulated, a cell function is inhibited. According to an example of the present invention, the BDB reduces the level of carbonylated protein generated by UVB.
In addition, according to an example of the present invention, it can be confirmed that the BDB is capable of protecting DNA in a cell from damage caused by UVB.
UVB radiation is a strong inducing agent of apoptosis, and produces ROS. From the following Example 8, it can be confirmed that cell death caused by apoptosis generated by UVB radiation is inhibited by decreasing the number of apoptotic body and DNS fragmentation when a BDB is treated.
In other words, the BDB having the aforementioned properties may be useful to inhibit skin damage caused by ultraviolet and protect skin from ultraviolet, so that the BDB may be used for a pharmaceutical composition for inhibiting and treating skin damage caused by ultraviolet, in which the pharmaceutical composition includes the BDB as an effective component. Further, the BDB compound itself has an ultraviolet absorption effect, so that the BDB can be used for a composition as an ultraviolet absorption composition including the BDB as an effective component.
Further, the 3-bromo-4,5-dihydroxybenzaldehyde (BDB) included in the composition according to the present invention as an effective component may be used in a type of salts, preferably pharmaceutically acceptable salts. Such salts may be preferably acid addition salts produced by pharmaceutically acceptable free acid, and examples of such a free acid may include an organic acid and inorganic acid. Examples of such an organic acid may include, but are not limited to, citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, methasulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, glutamic acid, and aspartic acid. Furthermore, examples of such an inorganic acid may include, but are not limited to, hydrochloric acid, bromic acid, sulfuric acid, and phosphoric acid.
The 3-bromo-4,5-dihydroxybenzaldehyde (BDB) compound according to the present invention may be naturally isolated, or may be produced by using a chemical synthetic method that is known in the prior art.
The composition according to the present invention including the BDB as an effective component may be a pharmaceutical composition.
The pharmaceutical composition according to the present invention may be prepared by using adjurvants that are pharmaceutically suitable and physiologically acceptable in addition to such an effective component. Examples of such adjurvants may include excipient, a disintegrating agent, a sweeting agent, a bonding agent, a coating agent, a blowing agent, a lubricant, a modifier, a flavouring agent, or the like.
The pharmaceutical composition may preferably be formulated by further including at least one pharmaceutically acceptable carrier in addition to the aforementioned effective component in order for an administration.
A formulation type of the pharmaceutical composition may be granules, powders, tablets, covered tablets, capsules, suppository, liquid formulations, syrups, juices, suspensions, an emulsion, medicinal drops, injectable liquid formulations, or the like. For example, in order to formulate in a type of tablets or capsules, an effective component may be bonded with an oral, nontoxic, pharmaceutically acceptable inert carrier, such as ethanol, glycerol, water, or the like. Further, in the case of need or necessary, a suitable bonding agent, lubricant, disintegrating agent and a color former may be included in a mixture. Examples of suitable bonding agent may include, but are not limited to, natural sugars, such as starch, gelatin, glucose, or beta-lactose, natural and synthetic gums, such as corn sweeting agents, acacia, tragacanth, or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Examples of the disintegrating agent may include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthane gum, and the like. In the composition formulated in a liquid solution, as a pharmaceutically acceptable carrier and a suitable material for sterilization and human body, saline solution, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and a mixture of at least one therefrom may be used, and if necessary, other general additives, such as antioxidants, a buffer solution, or bacteristat maybe included. Furthermore, it may be formulated in tables, granules, capsules, pills, or injectable tablets such as aqueous solution, suspensions, and emulsions by additionally adding diluent, a dispersing agent, surfactant, a bonding agent, and a lubricant. Further, it may preferably be formulated according to all the diseases or components by using the method as disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as a proper method in the prior art.
According to an example of the present invention, a pharmaceutically effective amount of the BDB according to the present invention may be 10 μM to 40 μM and preferably 30 μM. However, the pharmaceutically effective amount may be properly changed according to a degree of skin damage, an age, a body weight, a health condition, sex, an administration route, a treatment period of a patient, and the like.
According to an example of the present invention, the pharmaceutical composition of the present invention may be a skin composition for external use. Further, the composition of the present invention has an ultraviolet absorption effect, so that it may be used as a skin composition for external use to be applied to skin, or sunscreen as a cosmetic composition for absorbing ultraviolet, and the like.
The pharmaceutically skin composition for external use according to the present invention may be prepared and used in a type of a pharmaceutically skin composition for external use, such as cream, gel, a patch, a spraying agent, ointment, a hardening agent, lotions, liniments, pastes, and cataplasma as a skin composition for external use having a skin protective effect from ultraviolet. However, the present invention is not limited thereto.
Further, the composition of the present invention may be a cosmetic composition for inhibiting skin damage caused by ultraviolet and protecting skin from ultraviolet, in which the composition includes a BDB as an effective component.
In a case in which the composition of the present invention is prepared in a cosmetic composition, the composition of the present invention may include components that are generally used for the cosmetic composition as well as the BDB as disclosed above, and for example, general adjurvants such as antioxidant, stabilizer, a solubilizing agent, vitamins, pigments, and flavouring, and a carrier.
Further, the composition of the present invention may be used by mixing organic sunscreen agents that have conventionally been used within the range, in which a skin protective effect is not damaged by reacting with a BDB, in addition to the BDB as disclosed above.
Examples of such organic sunscreen agents may include at least one selected from the group consisting of glyceryl PABA, drometrizole trisiloxane, drometrizole, digalotrioleate, disodiumphenylbenzimidazoletetrasulfonate, diethylhexylbutamidotriazone, diethylaminohydroxybenzoylhexylbenzoate, DEA-methoxycinnamate, a mixture of Lowsone and dihydroxyacetone, methylene bis-benzotriazolyl tetramethylbutylphenol, 4-methylbenzylidene camphor, menthyl anthranilate, benzophenone-3(oxybenzone), benzophenone-4, benzophenone-8(dioxypebenzone), butylmethoxydibenzoylmethane, bisethylhexyloxyphenolmethoxyphenyltriazine, cinoxate, ethyldihydroxypropyl PABA, octocrylate, ethylhexyldimethyl PABA, ethylhexylmethoxycinnamate, ethylhexyl salicylate, ethylhexyl triazone, isoamyl-p-methoxycinnamate, polysilicone-15 (dimethicodiethylbenzalmalonate), terephthalylidene dicamphor sulfonic acid, salts thereof,
TEA-salicylate, and aminobenzoic acid (PABA).
Products that can use the cosmetic composition of the present invention may include cosmetic products, such as an astringent, skin lotion, nutrition lotion, all kinds of creams, essences, packs, and foundations, cleansing, face cleansing products, soaps, treatments, cosmetic solutions, and the like.
A specific formulation of the cosmetic composition according to the present invention includes skin lotions, skin softeners, skin toners, astringent, lotions, milk lotions, moisture lotions, nutrition lotions, massage creams, nutrition creams, moisture creams, hand creams, essences, nutrition essences, packs, soaps, shampoos, cleansing foams, cleansing lotions, cleansing creams, body lotions, body cleansers, an emulsion, lipsticks, make-up bases, foundations, press powders, loose powders, eye shadows, and the like.
According to a preferable embodiment of the present invention, a content of the BDB of the present invention is 10 μM to 40 μM and preferably 30 μM relative to the total weight of the composition. When the content of the BDB is less than 10 μM, an ultraviolet absorption effect maybe greatly decreased. On the other hand, when it exceeds 40 μM, skin irritation may be caused, and also a dosage form problem may be caused.
Meanwhile, the cosmetic composition according to the present invention may be formulated by including the BDB inside nano-liposome, and then stabilizing the BDB. When the compound is included inside the nano-liposome, the compound is stabilized, so that problems such as precipitation, discolorization, and a smell change may be solved, and a percutaneous absorption rate and solubility of the component maybe increased when formulating into a dosage form. Therefore, effectiveness to be expected from the compound may be maximally exhibited.
The nano-liposome used in the present invention means liposome having an average particle diameter of 10 to 500 nm with a type of a general liposome. According to a preferable embodiment of the present invention, an average particle diameter of the nano-liposome is 50 to 300 nm. When the average particle diameter of the nano-liposome exceeds 300 nm, among the technical effects to be achieved in the present invention, an improvement of dermal penetration and an improvement of dosage form stability may be very weak. The nano-liposome used for stabilizing the BDB compound according to the present invention may be prepared by a mixture including polyol, an oil component, surfactant, phospholipid, fatty acid, and water.
The polyol used in the nano-liposome of the present invention includes, but is not limited to, preferably, at least one selected from the group consisting of propylene glycol, dipropylene glycol, 1,3-butylene glycol, glycerin, methyl propanediol, isopropylene glycol, pentylene glycol, erythritol, xylitol, sorbitol, and mixtures thereof. The used amount thereof is 10 to 80 wt % and preferably 30 to 70 wt % relative to the total weight of the nano-liposome.
The oil component used for preparing the nano-liposome of the present invention may include various oils that are known in the prior art, but preferably hydrocarbon-based oils such as hexadecane and paraffin oils, silicone oils such as ester-based synthetic oil, dimethicone and cycliomethicone-based oils, animal and vegetable oils such as sunflower oil, corn oil, soybean oil, avocado oil, sesame seed oil, and fish oil, sphingoid liqid such as ethoxylated alkylether-based oil, propoxylated alkyleter-based oil, phytosphingosine, sphingosine, and sphinganine, cerebroside cholesterol, cytosterol cholesteryl sulfate, cytosteryl sulfate, C10 to C40 fatty alcohol and mixtures thereof. The used amount thereof may be 1.0 to 30.0 wt % and preferably 3.0 to 20.0 wt % relative to the total weight of the nano-liposome.
The surfactant used for preparing the nano-liposome of the present invention may include any things that are known in the prior art. Examples thereof may include anionic surfactant, cationic surfactant, ampholytic surfactant, and nonionic surfactant. Preferably, anionic surfactant and nonionic surfactant may be used. Specific examples of the anionic surfactant may include alkylacylglutamate, alkyl phosphate, alkyl lactylate, dialkyl phosphate, and trialkyl phosphate. Specific examples of the nonionic surfactant may include alkoxylated alkyl ether, alkoxylated alkyl ester, alkylpolyglycoside, polyglyceryl ester, and sugar ester. Still most preferable examples of the surfactant may include polysorbates belonging to nonionic surfactant. The used amount thereof may be 0.1 to 10 wt % and preferably 0.5 to 5.0 wt % relative to the total weight of the nano-liposome.
The phospholipid that is another component used for preparing the nano-liposome of the present invention may be ampiphilic lipid, and examples thereof may include natural phospholipid (for example, egg yolk lecithin or soybean lecithin, sphingo myelin) and synthetic phospholipid (for example, dipalmitoyl phosphatidylcholine or hydrogenated lecithin), and preferably lecithin. Especially, unsaturated lecithin or saturated lecithin that is naturally derived and extracted from a soybean or the yolk of an egg is preferable. In general, in natural lecithin, the amount of phosphatidylcholine is 23 to 95% and the amount of phosphatidylethanolamine is 20% or less. In a preparation of the nano-liposome of the present invention, the used amount of the phospholipid is 0.5 to 20.0 wt % and preferably 2.0 to 8.0 wt % relative to the total weight of the nano-liposome.
The fatty acid used for preparing the nano-liposome of the present invention is higher fatty acid, and preferably includes saturated or unsaturated fatty acid of C12 to C22 alkyl chains, such as lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, and linoleic acid. The used amount thereof may be 0.05 to 3.0 wt % and preferably 0.1 to 1.0 wt % relative to the total weight of the nano-liposome.
The water used for preparing the nano-liposome of the present invention may generally be deionized distilled water, and the used amount thereof may be 5.0 to 40 wt % relative to the total weight of the nano-liposome.
The preparation of the nano-liposome may be achieved through various methods that are known in the prior art, and most preferably, the nano-liposome may be prepared by applying the mixture including the aforementioned components to a high pressure homogenizer. The preparation of the nano-liposome using the high pressure homogenizer may be performed under various conditions (for example, a pressure, the number of performances, and the like) according to the desired particle size, and preferably the nano-liposome may be prepared by passing the mixture through the high pressure homogenizer one to five times under pressure of 600 to 1200 bar.
The cosmetic composition for protecting skin according to the present invention may include the BDB in an amount of 10 to 40 μM and preferably 30 μM relative to the total weight of the nano-liposome in order to stabilize a dosage form.
Hereinafter, the present invention will be described in more detail with reference to Examples. However, those
Examples are only for illustrating the present invention in more detail, but the range of the present invention is not intended to be limited to those Examples.
Statistical Analysis
All measurements were performed in triplicate, and all values were expressed as the mean ±the standard error. The results were subjected to an analysis of variance (ANOVA) using the Tukey's test to analyze differences between means. In each case, a P value of <0.05 was considered statistically significant.
Reagents
3-Bromo-4,5-dihydroxylbenzaldehyde (BDB, Matrix Scientific, Columbia, S.C., USA), N-acetyl cysteine (NAC), 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA), [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium] bromide (MTT) and Hoechst 33342 dye were purchased from Sigma Chemical Company (St. Louis, Mo., USA). All other chemicals and reagents were of analytical grade.
Cell culture
Human keratinocytes (HaCaT cells) were obtained from the Amore Pacific Company (Gyeonggi-do, Republic of Korea). Cells were maintained at 37° C. in an incubator with a humidified atmosphere of 5% CO2. Cells were cultured in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal calf serum, streptomycin (100 μg/mL) and penicillin (100 unit/mL).
BDB Effectiveness on UVB-Induced Apoptosis
The effect of BDB on the viability of HaCaT cells was assessed as follows. Cells were seeded in a 96-well plate at a density of 1×105 cells/mL. Sixteen hours after plating, BDB was added at a concentration of at 10, 20, 30, 40, and 50 μM. To evaluate the ability of BDB to protect keratinocytes against UVB-exposure, BDB was added at a concentration of 10, 20 and 30 μM, and cells were exposed to UVB radiation one hour later and incubated at 37° C. for 24 h. Fifty microliter of MTT stock solution (2 mg/mL) was added to each well to yield a total reaction volume of 200 μl. After incubating the cells for 4 h, the plate was centrifuged at 800×g for 5 min, and the supernatants were aspirated. The formazan crystals in each well were dissolved in dimethylsulfoxide (150 μl), and the absorbance at 540 nm was read on a scanning multi-well spectrophotometer.
As a result, the BDB does not exhibit toxicity on human HaCaT keratinocyte up to the concentration of 30 μM (see
Free Radical Removal Ability of BDB
DPPH radical removal ability and intracellular reactive oxygen species (ROS) removal ability were measured in order to confirm free radical removal ability of the BDB of the present invention prepared from Example.
<3-1> Measurement of DPPH Radical Removal Ability
BDB at a concentration of 10, 20, 30 μM and 2 mM NAC were added to a 1×10−4 M solution of DPPH in methanol. The resulting reaction mixture was shaken vigorously. After 3 h, the amount of unreacted DPPH was measured at 520 nm using a spectrophotometer.
As a result, the BDB scavenged DPPH radical depending on the volume. As illustrated in a black bar in
<3-2> Measurement of Intracellular Reactive Oxygen Species (ROS) Removal Ability
The DCF-DA method was used to detect intracellular ROS levels in HaCaT keratinocytes generated by either H2O2 or UVB radiation. For the detection of ROS in H2O2-treated cells, cells were seeded at a density of 1.5×105 cells/well. Sixteen hours after plating, cells were treated with BDB at a concentration of 10, 20, 30 μM and 2 mM NAC. After 30 min, H2O2 (1 mM) was added to the plate. Cells were incubated for an additional 30 min at 37° C., and DCF-DA solution (25 μM) was then added. Ten minutes after the addition of DCF-DA, the fluorescence of 2′,7′-dichlorofluorescein (DCF) was detected and quantified using a PerkinElmer LS-5B spectrofluorometer (PerkinElmer, Waltham, Mass., USA). For the detection of ROS in UVB-exposed cells, cells were treated with BDB as above. After one hour, cells were exposed to UVB radiation at a dose of 30 mJ/cm2. The UVB source was a CL-1000M UV Crosslinker (UVP, Upland, Calif., USA), which was used to deliver an energy spectrum of rays (280 to 320 nm). Cells were incubated for an additional 24 h at 37° C., DCF-DA solution (25 μM) was added and detected as above.
As a result, it was confirmed that the BDB removes an intracellular ROS induced by H2O2. As illustrated with alight gray bar in
<3-3> Detection of Superoxide Anion
The superoxide anion was produced via the xanthine/xanthine oxidase system and then reacted with a nitrone spin trap, DMPO. The DMPO/.OOH adducts were detected using a JES-FA electron spin resonance (ESR) spectrometer (JEOL, Tokyo, Japan). Briefly, ESR signaling was recorded 5 min after 20 μl of xanthine oxidase (0.25 U/mL) was mixed with 20 μl each of xanthine (5 mM), DMPO (1.5 M) and BDB (30 μM). The ESR spectrometer parameters were set at a magnetic field of 336 mT, power of 1.00 mW, frequency of 9.4380 GHz, modulation amplitude of 0.2 mT, gain of 500, scan time of 0.5 min, scan width of 10 mT, time constant of 0.03 sec, and temperature of 25° C.
As the result of analyzing a removal effect of the BDB on the superoxide anion and hydroxyl radical by using an ESR spectrometry, as illustrated in
<3-4> Detection of Hydroxyl Radical
The hydroxyl radical was generated by the Fenton reaction (H2O2+FeSO4) and then reacted with DMPO. The resultant DMPO/.OH adducts were detected using an ESR spectrometer. The ESR spectrum was recorded 2.5 min after a phosphate buffer solution (pH 7.4) was mixed with 0.2 mL each of 0.3 M DMPO, 10 mM FeSO4, 10 mM H2O2, and BDB (30 μM). The ESR spectrometer parameters were set at a magnetic field of 336 mT, power of 1.00 mW, frequency of 9.4380 GHz, modulation amplitude of 0.2 mT, gain of 200, scan time of 0.5 min, scan width of 10 mT, time constant of 0.03 sec, and temperature of 25° C.
As a result, as illustrated in
UVB Absorption Analysis
To study the UVB absorption spectra of BDB, which was diluted in DMSO at a ratio 1:500 (v/v), it was scanned by UV at 200-500 nm using Biochrom Libra S22 ultraviolet/visible spectrophotometer.
As a result, as illustrated in
Lipid Peroxidation Inhibition Effect by BDB
A cell was exposed to UVB, and then after 24 hours, effects of BDB in inhibiting the cell membrane lipid peroxidation, a protein modification, and a cell DNA damage of UVB-irradiated cell was observed. Lipid peroxidation was assayed by the determination of 8-isoprostane levels in the culture medium. A commercial enzyme immunoassay (Cayman Chemical, Ann Arbor, Mich., USA) was employed according to the manufacturer's instructions. Lipid peroxidation was also estimated using a fluorescent probe, DPPP. Cells were incubated with 5 μM. DPPP for 15 min in the dark and then exposed to UVB. DPPP fluorescence image was captured using a Zeiss Axiovert 200 inverted microscope at an excitation wavelength of 351 nm and an emission wavelength of 380 nm and quantified.
As a result, as illustrated in
Protein Carbonyl Formation Inhibition Effect by BDB
The amount of carbonyl formation in protein was determined using an Oxiselect™ protein carbonyl ELISA kit purchased from Cell Biolabs (San Diego, Calif., USA) according to the manufacturer's instructions.
As a result, as illustrated in
Protective Effect of BDB from DNA Damage
The degree of oxidative DNA damage was determined in a comet assay. Cell suspension was mixed with 75 μL of 0.5% low melting agarose (LMA) at 39° C. and the mixture was spread on a fully frosted microscopic slide pre-coated with 200 μL of 1% normal melting agarose (NMA). After solidification of the agarose, the slide was covered with another 75 μl of 0.5% LMA and then immersed in a lysis solution (2.5 M NaCl, 100 mM Na-EDTA, 10 mM Tris, 1% Trion X-100 and 10% DMSO, pH 10) for 1 h at 4° C. The slides were then placed in a gel-electrophoresis apparatus containing 300 mM NaOH and 10 mM Na-EDTA (pH 13) for 40 min to allow for DNA unwinding and the expression of the alkali-labile damage. An electrical field was then applied (300 mA, 25 V) for 20 min at 4° C. to draw the negatively charged DNA towards the anode. The slides were washed three times for 5 min at 4° C. in a neutralizing buffer (0.4 M Tris, pH 7.5), stained with 75 μL of propidium iodide (20 μg/mL) and observed using a fluorescence microscope and an image analyzer (Kinetic Imaging, Komet 5.5, UK). The percentage of total fluorescence in the DNA tails and the tail length of 50 cells per slide were recorded.
When a cell was exposed to UVB, the length and ratio of DNA tail to the cell tail were increased. When the cell was exposed to UVB, the ratio of DNA in the tail was increased to be 37%. In addition, as a result of treating with the BDB, as illustrated in
Effect on Apoptosis Induced by UVB Irradiation
A direct relationship to apoptosis induced by UVB irradiation was investigated, and inhibition ability of BDB on apoptosis induced by UVB was investigated.
<8-1> Nuclear Staining with Hoechst 33342
Cells were treated with BDB at a concentration of 30 μM and exposed to UVB radiation 1 h later. Cells were incubated for an additional 24 h at 37° C. Hoechst 33342 (1.5 μL of a 10 mg/mL stock), a DNA-specific fluorescent dye, was added to each well, and the cells were incubated for 10 min at 37° C. The stained cells were visualized under a fluorescence microscope equipped with a CoolSNAP-Pro color digital camera. The degree of nuclear condensation was evaluated, and the apoptotic cells were quantified. As a result, as illustrated in
<8-2> Sub-G1 Hypodiploid Cells
Flow cytometry was performed in order to determine the apoptotic sub-G1 hypodiploid cells. Cells were treated with BDB at a concentration of 30 μM and exposed to UVB radiation 1 h later. Cells were incubated for an additional 24 h at 37° C. Cells were harvested, and fixed in 1 mL of 70% ethanol for 30 min at 4° C. Cells were washed twice with PBS, and then incubated for 30 min in the dark at 37° C. in 1 mL of PBS containing 100 μg propidium iodide and 100 μg RNase A. Flow cytometric analysis was performed using a FACS Calibur flow cytometer (Becton Dickinson, Mountain View, Calif., USA). Sub-G1 hypodiploid cells were assessed based on the histograms generated using the computer programs, Cell Quest and Mod-Fit.
In addition to a morphological evaluation, a protective effect of BDB on apoptosis can be confirmed by a flow cytometry. As a result of analyzing DNA in cells exposed to UVB, as illustrated in
<8-3> DNA Fragmentation
Cellular DNA fragmentation was assessed by analyzing the extent of cytoplasmic histone-associated DNA fragmentation using a kit from Roche Diagnostics (Portland, Oreg., USA) according to the manufacturer's instructions. A cytoplasmic histone-associated DNA fragmentation was increased in UVB-irradiated cells as compared with control cells. The level of DNA fragmentation was decreased in UVB-irradiated cells that were treated with BDB (see
Accordingly, from the aforementioned results, the inventors of the present invention can found that the BDB effectively inhibits apoptosis confirmed by experiments of a degree of nuclear condensation and DNA fragmentation caused by ultraviolet irradiation; has ultraviolet absorption ability and free radical removal ability; inhibits a lipid peroxidation and protein carbonyl formation; and protects DNA damage; and thereby ultimately has excellent effect in protecting a cell from ultraviolet.
As set forth above, according to exemplary embodiments of the invention, the composition including a 3-bromo-4,5-dihydroxybenzaldehyde (BDB) has a photo-protective effect to cell damage induced by ultraviolet in human skin keratinocyte, and a free radical and reactive oxygen species-removal activities. Therefore, the BDB reduces apoptosis and recoveries cell viability, so that the BDB exhibits antioxidant activity, protects cell damage from ultraviolet, and also has an ultraviolet absorption effect. Thus, the BDB of the present invention can be usefully used as a raw material for a functional cosmetic composition or a pharmaceutical composition for protecting skin cell from ultraviolet.
While the present invention has been shown and described in connection with the exemplary embodiments, it will be apparent to those skilled in the art that modifications and variations can be made without departing from the spirit and scope of the invention as defined by the appended claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2012-0069710 | Jun 2012 | KR | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13846191 | Mar 2013 | US |
| Child | 15890234 | US |
