KR20150019708A - UV protecting cosmetic composition comprising ginsenoside Rg3 - Google Patents

Abstract

The present invention relates to a cosmetic composition for protecting ultraviolet rays comprising ginsenoside-Rg3 as an active ingredient. More particularly, the present invention relates to a cosmetic composition for protecting ultraviolet rays comprising 20 (S) -ginchenoside-Rg3 or 20 (R) As an active ingredient, and cosmetic compositions of the present invention are applied to human skin to protect skin from ultraviolet rays. 20 (S) -gincenoside-Rg3, an active ingredient of the cosmetic composition according to the present invention, can effectively inhibit the generation of reactive oxygen species (ROS) induced in skin cells upon irradiation with ultraviolet rays, The ginsenoside-Rg3 and 20 (R) -ginsenoside-Rg3 are excellent in the activity of suppressing the activity and expression of MMP-2, which is expressed and activated in skin cells upon irradiation with ultraviolet rays, It can be utilized as a material for cosmetics.

Description

(UV protecting cosmetic composition containing ginsenoside Rg3) containing ginsenoside-Rg3 as an active ingredient,

The present invention relates to a cosmetic composition for UV-screening comprising ginsenoside-Rg3 as an active ingredient.

In human skin, various physical and chemical changes occur in the aging process. The cause of this phenomenon is divided into intrinsic aging and photo-aging. Free radicals can be caused by activation of ultraviolet rays, stress, disease state, environmental factors, wounds, and aging. If such state is deepened, the antioxidant defense network existing in the living body is destroyed, And aging.

In particular, when ultraviolet rays contained in sunlight are directly irradiated to the skin, it promotes erythema formation and formation of melanin pigment in skin cells, which may be a cause of stain and dullness, and react with sebum secreted from the epidermis, It can cause skin troubles by generating, and can cause skin cancer if it is severe. Ultraviolet rays are classified into UV-A (320 to 400 nm), UV-B (280 to 320 nm) and UV-C (200 to 280 nm) depending on the wavelength. Among them, ultraviolet Are known as UV-A and UV-B.

In addition, UVB exposure increases the formation of free radicals and reactive oxygen species (ROS) in the skin and induces oxidative stress on cellular components such as DNA, cell membrane and protein, It is known to cause aging. For this reason, various studies are currently under way to prevent UVB-induced skin damage of natural antioxidants (Joshi et al., 2012; Gonzales-Castaeda et al., 2011; Petrova et al., 2011; , 2010).

On the other hand, it is known that ginseng has a useful effect in promoting human health as a well-known herb worldwide. It is known that ginseng has anticancer activity, anti-allergy, anti-diabetic effect and pain relief.

In addition, ginseng contains active ingredients such as ginsenosides, polysaccharides, peptides, polyacetylenic alcohols and fatty acids. In particular, there are more than 30 kinds of ginsenosides, and ginsenosides Is recognized as a major component of various pharmacological efficacy.

Ginsenoside is a so-called ginseng saponin, which is classified into three classes based on the aglycon residue. (Re, Rg1, Rg2, Rf, Rh1, etc.), the second is the protopanaxyldiol (Ra1, Ra2, Ra3, Rb1, Rb2, Rb3, Rc, Is classified as oleanolic (Ro) ginsenoside.

Ginseng saponin has been reported to have different pharmacological effects depending on the type of sugar bound to the aglycone, the number of bound sugars or the binding site (Lee, SR, et al., Korean J. Ginseng Sci. Pharm Res., 27: 1136- 1140, 2006), for example, in the case of a microorganism, for example, , Gene to ginsenoside Rh2 is present the two kinds of diastereomers, 20 (R) - ginsenoside -Rh2 [20 (R) -Rh2] and 20 (S) - ginsenoside -Rh2 [20 (S) - Rh2].

The effect of 20 ( R ) -ginsenoside-Rh2 [20 ( R ) -Rh2] and 20 ( S ) -gincenoside-Rh2 [20 ( S ) -Rh2] on the therapeutic effect of osteoporosis was investigated. R) bar only -Rh2 found that the treatment of osteoporosis and effects (Liu etal., 2009), while the 20 (S) than the 20 (R) -Rh2 for anticancer activity -Rh2 cycle inhibiting proliferation of cancer cells, and cell (Zhang et al ., 2011). It has been found that the G1 phase is arrested and has excellent anticancer activity.

Therefore, it is necessary to search for new substances having major physiological activities for ginseng, which is a medicinal plant with various physiological activities. In addition, it is necessary to investigate the exact correlation between these structures and physiological activities for the stereoisomers of ginsenosides having different structures Is required.

The ginsenoside Rg3 selected by the present inventors is known to exhibit the effects of inhibiting cancer cell metastasis, inhibiting platelet aggregation, antithrombotic action, hepatic injury-inhibiting action, vasodilatory action, and anticancer action (see Min 20 (S) -glycinoside Rg3 and 20 (R), which are two isomers of ginsenoside Rg3, have been reported in the literature (JK et al., Biochem. Biophys. Res. Commun., 349: 987-994, - Studies on the ultraviolet blocking effect of ginsenoside Rg3 have not been disclosed.

Thus, the present inventors have completed the present invention by determining how 20 (S) -ginchenoside Rg3 and 20 (R) -ginnenoside Rg3 each have an ultraviolet blocking effect.

Korean Patent Application 2006-0055001A

Therefore, an object of the present invention is to provide a cosmetic composition for UV-screening which comprises ginsenoside-Rg3 as an active ingredient.

Another object of the present invention is to provide a cosmetic method characterized by having an effect of protecting the skin from ultraviolet rays by applying the cosmetic composition of the present invention to human skin.

In order to achieve the above object, the present invention provides a cosmetic composition for protecting ultraviolet rays comprising ginsenoside-Rg3 as an active ingredient.

In one embodiment of the present invention, the ginsenoside-Rg3 may be 20 (S) -ginchenoside-Rg3 or 20 (R) -ginnenoside-Rg3.

In one embodiment of the present invention, the ginsenoside-Rg3 may be contained in the composition at a concentration of 1 to 30 uM.

In one embodiment of the present invention, the 20 (S) -ginsenoside-Rg3 may have an activity of inhibiting the production of reactive oxygen species (ROS) generated by ultraviolet irradiation.

In one embodiment of the present invention, 20 (S) -gincenoside-Rg3 and 20 (R) -gencenoside-Rg3 have activity of MMP-2 (matrix metalloproteinases-2) . ≪ / RTI >

In one embodiment of the present invention, the ultraviolet ray may be UV-B having a wavelength of 280 to 320 nm.

In one embodiment of the invention, the composition is in the form of a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, cleansing oil, powder foundation, emulsion foundation, wax foundation, Spray, and the like.

The present invention also provides a cosmetic method characterized by having the effect of protecting the skin from ultraviolet rays by applying the cosmetic composition of the present invention to human skin.

The present invention relates to a cosmetic composition for protecting ultraviolet rays comprising 20 (S) -gincenoside-Rg3 or 20 (R) -ginnenoside-Rg3 as an active ingredient and a cosmetic composition of the present invention, The present invention relates to a make-up method characterized by having an effect of protecting the skin. 20 (S) -gincenoside-Rg3, an active ingredient of the cosmetic composition according to the present invention, can effectively inhibit the generation of reactive oxygen species (ROS) induced in skin cells upon irradiation with ultraviolet rays, The ginsenoside-Rg3 and 20 (R) -ginsenoside-Rg3 are excellent in the activity of suppressing the activity and expression of MMP-2, which is expressed and activated in skin cells upon irradiation with ultraviolet rays, It can be utilized as a material for cosmetics.

Figure 1 shows the structural formulas of 20 (S) -ginsenoside-Rg3 and 20 (R) -gencenoside-Rg3, respectively.
FIG. 2 shows the activity (a), the level of nitric oxide (b) and the cell viability (c) of 20 (S) -gincenoside-Rg3 in HaCat cells when irradiated with HaCat cells .
FIG. 3 shows the activity (a), the level of nitric oxide (b) and the cell viability (c) of 20 (R) -ginnenoside-Rg3 in HaCat cells when irradiated with ultraviolet light in HaCat cells .
FIG. 4 shows the results of observing the amount of reactive oxygen species generated in the cells after treatment of ginsenoside-Rg3 with HaCat cells by concentration and ultraviolet ray B, S) -ginchenoside-Rg3, and 4b is the result of treating 20 (R) -gencenoside-Rg3.
FIG. 5 is a graph showing the results of treatment of 20 (S) -ginchenoside-Rg3 and 20 (R) -ginnenoside-Rg3 in human skin fibroblast cell line CCD986 and irradiating ultraviolet ray B, (5a and 5b) scavenging activity and cytotoxicity (4c and 4d).
FIG. 6 shows gelatin melting activity of MMP-1 and MMP-2 in HaCat cells after treatment with ginsenoside-Rg3 at different concentrations. The result of gelatin zymography analysis of 6A was 20 (S) - Ginsenoside-Rg3, and 6b is the result of treating 20 (R) -gencenoside-Rg3.
FIG. 7 shows Western blot analysis of inhibition of MMP-2 protein expression by treatment with 20 (S) -gencenoside-Rg3 in HaCat cells at various concentrations.

The inventors of the present invention have been studying new cosmetic materials capable of effectively protecting skin from ultraviolet rays, and have found that 20 (S) -ginceno, which is two structural isomers of ginsenoside-Rg3 derived from ginseng, specifically, ginsenoside- Side-Rg3 and 20 (R) -ginsenoside-Rg3, the present invention was completed.

Accordingly, the present invention is characterized by providing a cosmetic composition for protecting ultraviolet rays comprising ginsenoside-Rg3 derived from ginseng as an active ingredient.

The ginsenoside-Rg3, which is an active ingredient contained in the cosmetic composition of the present invention, is a compound having two structural isomers represented by the following structural formulas: 20 (S) -ginnenoside-Rg3 and 20 (R) Respectively.

The structural isomeric compounds of the two ginsenosides-Rg3 can be extracted and separated from ginseng, or synthesized by chemical synthesis.

As previously described in the prior art, ginseng contains various ginseng saponin components. In the present invention, the activity and mechanism of the ultraviolet blocking effect of each of the two stereoisomers of ginsenoside-Rg3 Respectively.

According to a number of recent studies, it has been reported that even though the compounds are structurally similar to each other, the chemical properties and physiological activity of the compounds due to their structural characteristics are completely different.

For example, as mentioned in the prior art, only 20 ( R ) -Rh2 has therapeutic effect on osteoporosis treatment effect and 20 ( S ) -Rh2 has insufficient therapeutic effect.

Thus, even if a particular compound has similar structural formulas such as stereoisomers, fine structural differences can exhibit completely different physiological activities, so that the function can not be deduced by the similarity of structures.

Meanwhile, in the present invention, various experiments were conducted to confirm whether 20 (S) -ginnenoside-Rg3 and 20 (R) -gencenoside-Rg3 can be used as functional cosmetics for ultraviolet screening, According to one embodiment of the present invention, HaCat cells as human keratinocyte cells and CCD986 cells as human dermal fibroblast cells were treated with 20 (S) -ginnenoside-Rg3 and 20 (R) -ginceno Side-Rg3, and ultraviolet ray B was irradiated, and then the amounts of active oxygen species and nitric oxide generated in each cell were measured.

As a result, 20 (S) -gincenoside-Rg3 showed activity to effectively inhibit active oxygen species generated by ultraviolet B irradiation in a concentration-dependent manner, while 20 (R) -gincenoside-Rg3 Showed little inhibitory effect on reactive oxygen species (see Figs. 2a and 3a).

In addition, as a result of analyzing the production amount of nitrogen oxide, it was found that the production amount of nitric oxide by ultraviolet irradiation was not influenced in all the groups treated with 20 (S) -ginenoside-Rg3 and 20 (R) -ginenoside-Rg3 (See FIGS. 2B and 3B).

Accordingly, the present inventors have found that only the structural isomer of 20 (S) -ginchenoside-Rg3 among ginsenosides-Rg3 can specifically inhibit active oxygen species generated by ultraviolet irradiation (particularly ultraviolet ray B) Lt; / RTI > activity.

In addition, UVB in ultraviolet rays promotes the production of reactive oxygen species and induces oxidative stress. Therefore, based on the above result that 20 (S) -gencenoside-Rg3 can effectively reduce ROS in UVB-irradiated cells, the present inventors have found that 20 (S) -ginnenoside-Rg3 is ultraviolet- -B can be used as a cosmetic material for blocking.

On the other hand, the analysis of the effect of ginsenoside-Rg3 on nitric oxide by ultraviolet light, unlike the results of reactive oxygen species inhibition, showed that both structural isomers had no effect on the production of nitrogen oxides (NO). That is, in the case of irradiating ultraviolet rays or not irradiating, the amount of similar nitrogen oxide was measured in all the groups treated with the concentration of ginsenoside-Rg3.

Nitric oxide (NO) acts as a physiological phenomenon, blood pressure control and neurotransmitter, and plays a pivotal role in the immune response. However, NO overexpressed by inducible NOS causes an inflammatory response and an abnormal immune system. In addition, when iNOS is induced by various stress inducers and abnormal accumulation of NO in the cells occurs, a reactive nitrogen species (RNS) is formed and the cells are killed.

However, according to the experimental results, it was found that the production of nitrogen oxides was not related to ultraviolet B irradiation. Thus, ultraviolet B is independent of the nitric oxide production mechanism, and 20 (S) -gincenoside-Rg3 has been shown to protect skin from ultraviolet rays through the mechanism of production of reactive oxygen species induced by ultraviolet B irradiation .

On the other hand, matrix metalloproteinases (MMPs) are zinc-ion-dependent proteolytic enzymes that degrade the extracellular matrix (ECM) present in the basement membrane or interstitial tissue (Steven D shapiro, Cell Biol, 10, 602, 1998). It is known that the activity of MMPs is regulated by gene expression, activation of inactive enzymes, and inhibition of enzyme activity (Woodhouse et al., Cancer, Nagase et al., J Biol Chem, 274, 21491, 1999).

These MMPs can be classified into five types depending on substrate specificity and basic structure, namely, collagenase (MMP-1, MMP-8, MMP-13) (MMP-2, MMP-9), stromelysin (MMP-3, MMP-7, MMP-10, MMP-11 and MMP-12), membrane type-MMPs , MT3-MMP, MT4-MMP) and unclassified MMPs (Kleiner DE et al., Cancer Chemother Pharmacol (1999) 43; 42-51).

The general structure of MMPs is composed of a signal peptide, a propeptide domain, a catalytic domain having a zinc (Zn) binding site, and a haemopexin-like domain linked to a catalytic domain by a hinge region ).

Among these various MMPs, gelatinase (MMP-2, MMP-9) is known to be continuously detected in malignant tissues and related to tumor aggressiveness and transit ability (Wilson CL et al., Proc Natl MMP-2 and MMP-9 are very similar enzymes at many receptors. In addition, MMP-2 and MMP-9 are very similar enzymes in many receptors. However, there is a clear difference in the regulation of these expression, glycosylation, activation of proenzyme and substrate. For example, MMP-2 is a 72-kDa unmalified protein whereas 92-kDa MMP-9 has two glycosylated sites in the prodomain and catalytic domains (LP Kotra et al. Biochemistry (1999) 38; 13937-13950, TS Mattu et al., Biochemistry (2000) 39; 15695-15704). Unlike MMP-2, which is always expressed and acting, MMP-9 is characterized by increased expression by stimulation of inflammatory mediators such as TNF- and is regulated by specific transcription factors such as NF-B (Sanseau et al J Biol Chem, 227, 35766, 2002). It is able to decompose most components of extracellular matrix and maintains physiological functions normally during the reconstruction of tissues such as wound healing fetal development, neovascularization, menstruation, ovulation, and pregnancy, but when over expressed, (Linda et al., Chem. Biol., 2, 466, 1998). It has been reported that rheumatoid arthritis, chronic ulcer, periodontitis, arteriosclerosis, growth of the stomach and invasion and metastasis of tumor cells.

In addition, these MMPs are known to increase their expression as collagen synthesis is reduced through complex signaling pathways in skin exposed to ultraviolet light (Lee et al., J. Dermatol Sci, 17, 182, 1998) It is known that MMP-1 is activated upon irradiation with ultraviolet light A, and activity of MMP-2 is increased upon irradiation with ultraviolet light B. It has been reported that an increase in MMP-2 activity in the skin caused by ultraviolet irradiation causes significant collapse of collagen in the skin, thereby affecting the collagen collapse of the dermal layer and playing a very important role in photoaging (Lee et al. J. Dermatol Sci, 17, 182, 1998).

Therefore, when collagen is reduced by MMP-2 and MMP-9, skin strength and tensile strength are lowered, and the function of protecting the skin is significantly reduced, thereby accelerating skin aging. Therefore, a material that inhibits the activity or expression of such MMP from ultraviolet rays can be used as a material capable of protecting the skin from ultraviolet rays.

Thus, the inventors of the present invention conducted studies to confirm whether 20 ( S ) -ginnenoside-Rg3 and 20 (R) -gencenoside-Rg3 of the present invention inhibit the activity or expression of MMPs (Matrix Metalloproteinase) , According to one embodiment of the present invention, when 20 ( S ) -ginnenoside-Rg3 and 20 (R) -gencenoside-Rg3 were treated in HaCat cells irradiated with ultraviolet B, MMP-2 , While activity of MMP-1 remained unchanged. In addition, the effect on the activity of MMP-2 was shown to be superior to 20 ( S ) -ginnenoside-Rg3 compared to 20 (R) -ginnenoside-Rg3 (see FIGS. 6 and 7).

Thus, these results show that both of the two structural isomers of ginsenoside-Rg3 have an activity of protecting skin cells by ultraviolet irradiation. In particular, 20 ( S ) -gincenoside-Rg3 has 20 (R) - ginsenoside-Rg3. ≪ / RTI >

In addition, the ginsenoside-Rg3 of the present invention does not cause cytotoxicity to skin cells and thus has cell stability.

In order to confirm this, the present inventors measured the degree of apoptosis by MTT assay after treating ginsenoside-Rg3 with concentrations of HaCat cells, a human keratinocyte cell, and CCD986, a human skin fibroblast cell, (0, 5, 12, and 30 uM) treated with different concentrations showed no significant difference in cell survival compared to the control group without ginsenoside-Rg3 treatment. Therefore, it can be seen from the results that the ginsenoside-Rg3 of the present invention does not show toxicity to skin cells, so that it can be safely used for the production of cosmetics applied to the skin.

Therefore, the present invention can provide a cosmetic composition for protecting ultraviolet rays comprising ginsenoside-Rg3 as an active ingredient, and the composition may have a function of protecting skin from ultraviolet rays.

The composition may contain ginsenoside-Rg3 at a concentration of 1 to 30 uM and the ginsenoside-Rg3 may be 20 (S) -ginnenoside-Rg3 or 20 (R) -ginnenoside- And preferably 20 (S) -glycinoside-Rg3.

In addition, if the composition according to the present invention contains ginsenoside-Rg3 in an amount of less than 1 uM, the desired effects described above can not be obtained. If the composition has an amount of more than 30 uM, unexpected side effects may occur in the skin, The degree of increase is insignificant. Therefore, it is preferable that the content is included in the range described above.

In addition, the composition according to the present invention has skin protection and ultraviolet shielding effect by ultraviolet ray B in ultraviolet rays. Generally, ultraviolet rays are divided into three wavelength regions of ultraviolet rays A, B and C.

First, UV-A (320 ~ 400nm) is not absorbed by the ozone layer, its wavelength range is 0.32 ~ 0.40, and its energy is less than that of UV-B, but the skin can be smoothed. UV-B is the main actor in burning skin, but UV-A not only makes skin tick, but also acts on skin's immune system and can cause long-term skin damage due to skin aging. Recently, studies have reported that the risk of developing skin cancer is the same as that of UV-B if the UV-A exposure time is long enough to smear the skin.

UV-B (280-320 nm) is mostly absorbed by the ozone layer, but some reach the surface. UV-B, which reaches the Earth in very small quantities, has a wavelength range of 0.28 to 0.32. It burns the skin of the animal, penetrates the skin tissue and sometimes causes skin cancer. Most of the causes of skin cancer are exposure to sunlight and UV- B is related to.

In addition, UV-C (100 to 280 nm) is completely absorbed by the ozone layer. Among ultraviolet rays with a wavelength range of 0.20 to 0.29, UV-C causes chromosomal aberrations, kills single-cell organisms, and damages the cornea of the eye. Fortunately, this range of ultraviolet light, known as UV-C, is almost all absorbed by stratospheric ozone.

UV protection for skin protection provided by the composition of the present invention corresponds to UV-B having a wavelength range of 280 to 320 nm among the three ultraviolet rays.

Further, the components contained in the cosmetic composition of the present invention may contain components commonly used in cosmetic compositions other than ginsenoside-Rg3 as an active ingredient, and examples thereof include antioxidants, stabilizers, solubilizers, vitamins, Such as conventional adjuvants, and carriers.

The cosmetic composition of the present invention can be prepared into any of the formulations conventionally produced in the art and can be used in the form of solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, , Oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto. More specifically, the cosmetic composition of the present invention can be manufactured in the form of a soft lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder.

When the formulation of the present invention is a paste, cream or gel, an animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier component .

When the formulation of the present invention is a solution or an emulsion, a solvent, a dissolving agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid esters.

In the case where the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In the case of a spray, in particular, / Propane or dimethyl ether.

When the formulation of the present invention is a surfactant-containing cleansing, the carrier component may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide ether Alkylamido betaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester.

In addition, the present invention can provide a cosmetic method which has an effect of protecting the skin from ultraviolet rays by using a cosmetic composition comprising the ginsenoside-Rg3 of the present invention as an active ingredient.

The cosmetic process of the present invention refers to all cosmetic processes using the cosmetic composition of the present invention. That is, all methods known in the art using a cosmetic composition belong to the cosmetic method of the present invention.

The cosmetic composition of the present invention may be used alone or in combination, or may be used in combination with other cosmetic compositions other than the present invention. The cosmetic composition according to the present invention may be used according to a conventional method of use, and may be used in a number of times depending on the skin condition or taste of the user.

When the cosmetic composition of the present invention is a soap, a surfactant-containing cleansing formulation or a surfactant-free cleansing formulation, it may be applied to the skin and then wiped off, washed or rinsed with water. The surfactant-containing cleansing formulation may be a cleansing foam, a cleansing water, a cleansing towel and a cleansing pack. The surfactant-free cleansing formulation may be a cleansing cream, , Cleansing lotion, cleansing water and cleansing gel, but is not limited thereto.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited to these embodiments.

< Example >

reagent

The reagents used for the following examples are as follows. First, 20 (S) -gincenoside Rg3 [20 (S) -Rg3, purity98%] and 20 (R) -ginenoside Rg3 [20 (R) ), Sodium nitrate, sodium dodecyl sulfate (SDS), 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide, and DCFH-DA (2,7-dichlorofluorescein diacetate) and Griess reagent were purchased from Sigma (St Louis, MO, USA). Also, DMEM (Dulbeccos modified Eagles medium), fetal bovine serum (FBS), penicillin-strepdomycin and trypsin-EDTA were obtained from HyCone Laboratories Inc. (Logan, UT, USA) Of the highest quality.

Cell culture

Human keratinocyte HaCat (ATCC, Manassas, Va., USA) and human skin fibroblast cell line CCD986 (ATCC, Manassas, Va., USA) were heat-treated with 10% FBS, 100 U / mL Penicillin and 100 g / mL streptomycin in a humidified atmosphere of 5% CO ₂ and at a temperature of 37 ° C.

UV -B ultraviolet treatment

The ultraviolet treatment in the following experiment was performed using an ultraviolet lamp (peak, 312 nm; model VL-6.M, Vilber Lourmat, Marine, France) as ultraviolet B (Model VLX-3W, Vilber Lourmat) instrument equipped with a spectrophotometer, 280 to 320 nm; model CX-312, Vilber Lourmat). The cultured cells were irradiated with 70 mJ / cm ² UV-B in each of the following experiments.

Statistical processing

Experimental results were expressed as mean standard deviation, and statistical comparisons of each experimental group were performed by unpaired Student's t-test. A p-value <0.05 was considered statistically significant.

< Example  1>

Gin Senocide - Rg3 of UV -B by investigation ROS  Production inhibition activity assay

The inventors of the present invention found that the ginsenoside Rg3 of the present invention, that is, 20 (S) -ginnenoside Rg3 and 20 (R) -ginnenoside Rg3 to ensure that the activity for erasing each ROS previously cultured human keratinocyte cell line (human keratinocyte cell) in HaCat (ATCC, Manassas, VA, USA) and CCD986 1.0 x 10 for ⁵ cells, respectively 5, 12 and 20 of 30 uM (S) - ginsenoside Rg3 and 20 (R) - after the pre-treatment by the ginsenoside Rg3 30 minutes before irradiation with ultraviolet light, it was examined ^{2 UV-B 70 mJ / cm} . To measure the ROS scavenging activity of the ginsenosides, a redox-sensitive fluorescent probe DCFH-DA, a method designed by Royall and Ischiropoulos, was used, that is, cells treated with the ginsenosides were treated with 10 uM in which process the DCFH-DA, and incubated at 37 ℃ for 30 minutes, then washed twice in medium without the in 1mL FBS, and then added back to the medium does not have a ^{1mL FBS, 70 mJ / cm 2} UV-B Respectively. Cells were collected and the amount of ROS in the cells was measured using a Multi-Mode Microplate Reader (Synergy ^™ Mx, BioTekInstruments, Winooki, VT, USA). The level of ROS was expressed as DCF-fluorescence.

As a result of the analysis, as shown in Figs. 2A and 3A, unusually, of the two isomers of ginsenoside Rg3, that is, 20 (S) -ginenoside Rg3, ultraviolet irradiation, particularly UV- (S) -ginsenoside Rg3 was treated with 20 (S) -gencenoside Rg3 compared with the case where only 20 (S) -ginsenoside Rg3 was not treated, , And active oxygen species were significantly reduced to 79.5%, 45.2%, and 43.2% at 5, 12, and 30 uM concentrations, respectively (see FIG. 2A). On the other hand, 20 (R) -ginsenoside Rg3 showed little activity to scavenge reactive oxygen species upon UV irradiation unlike 20 (S) -ginsenoside Rg3 (see FIG. 3A).

These results indicate that 20 (S) -ginenoside Rg3 is also active in the cleavage of active oxygen species in the case of CCD986 cells, a human skin fibroblast, similar to the results of FIGS. 2 and 3 for HaCat cells (See Figs. 5A and 5B).

The present inventors also observed the degree of ROS production of HaCat cells treated with 20 (S) -gincenoside Rg3 and 20 (R) -gencenoside Rg3 respectively and irradiated with ultraviolet rays through a confocal microscope, that is, 3 x 10 ⁵ HaCat cells were each divided into 6 wells, cultured overnight, washed twice with cold 1 ml of PBS, and then added with 1 ml of serum-free medium. Subsequent to the 20 (S) -Rg3, or 20 (R) -Rg3 and 10uM added to DCFH-DA 30 bun dongan 37 ^o C in the culture and the cells of 70mJ / cm ² After UV-banding, cells were observed with a Confocal Laser Scanning Microscope (Fluoview-FV300, Olympus, Tokyo, Japan) and the analysis was repeated at least three times.

As shown in FIG. 4, the 20 (S) -ginnenoside Rg3 showed a gradual decrease in fluorescence intensity in a concentration-dependent manner, and production of active oxygen species was inhibited (see FIG. 4A) (R) -ginsenoside Rg3 treated group showed continuous production of active oxygen species regardless of treatment concentration (see FIG. 4B).

Therefore, the inventors of the present invention found that the inhibition of the production of intracellular reactive oxygen species induced by ultraviolet light during the irradiation of ultraviolet light among the two isomers of ginsenoside Rg3 is not 20 (S) - ginsenoside Rg3, It was confirmed that this is due to ginsenoside Rg3.

< Example  2>

Gin Senocide - Rg3 of UV Analysis of inhibitory activity of nitric oxide formation by -B irradiation

The present inventors also investigated the effect of 20 (S) -gincenoside Rg3 and 20 (R) -gencenoside Rg3 on nitric oxide produced in skin cells due to UV-B irradiation. In Example 1 The amount of nitric oxide was measured after the ginsenosides were treated at each concentration and cell supernatants were obtained from UV-B irradiated cells.

Nitric oxide was measured by a colorimetric assay using the Griess reaction (Sherman et al., 1993) for cultured cells. More specifically, Griess reagent (1 % sulfanilamide-0.1% N- 1-naphthyl-ethylenediamine dihydrochloride in 2.5% phosphoricacid) were mixed with the culture medium and reacted at room temperature for 10 minutes. Then, the absorbance at 550 nm was measured by ELISA analysis. At this time, the standard curve was prepared using the known concentration of sodium nitrite (0 to 160 uM).

As a result of the analysis, as shown in Figs. 2B and 3B, the production of nitrogen oxides was found to be constant regardless of the irradiation with ultraviolet rays, and a certain level was also measured regardless of the amount of treatment with ginsenoside Rg3 .

These results indicate that the production of nitric oxide in skin cells is not related to ultraviolet irradiation, especially UV-B irradiation.

< Example  3>

Measurement of cytotoxicity

The present inventors conducted MTT analysis to confirm that 20 (S) -ginnenoside Rg3 and 20 (R) -ginnenoside Rg3 used in the present invention cause toxicity (effect on cell survival) to skin cell lines Respectively. For this purpose, 1.0 x 10 ⁵ cells of HaCat and CCD986 were each plated in 24-well plates, incubated overnight at 37 ^° C, washed twice with 1 ml of PBS, and then 1 ml of serum-free medium was added. Then 20 ( S ) -Rg3 or 20 ( R ) -Rg3 were each treated for 30 minutes and irradiated with ultraviolet light in the same manner as described above. After the medium was removed, the cells were reacted with 5 μg / ml MTT solution, and the cells were dissolved with DMSO and fixed with formalin. The degree of reduction of MTT was analyzed at 540 nm absorbance.

The analysis results, the survival of the cells, in HaCat cells in the above described conditions carried out in this experiment, that is, binary throughput of ginsenosides (to a concentration of 30uM), and ultraviolet radiation (70mJ / cm ²⁾ strength, as shown in Fig. 2c and 3c And the CCD986 cell line also had no significant effect on the survival of the cells (see FIGS. 5c and 5d).

< Example  4>

By ultraviolet irradiation MMPs On the expression and activity of Gin Senocide - Rg3 Impact Analysis

Matrix metalloproteinases (MMPs), which are substrate proteases, are known to increase expression upon irradiation with ultraviolet light. They inhibit the biosynthesis of proto-collagen to reduce the content of collagen, thereby promoting wrinkle formation of the skin and causing pigmentation, It makes it dull.

Thus, the present inventors have investigated the effect of 20 (S) -gincenoside Rg3 and 20 (R) -gencenoside Rg3 on MMPs, particularly MMP1 and MMP2, which are increased in expression upon UV irradiation, using gelatin zymography and Western blot.

To this end, a culture solution of cells treated and cultured under the same conditions as in Example 1 was first obtained, and then the cells were cultured in a 10% (v / v) SDS-PAGE gel impregnated with 1 mg / The proteins in the gel were denatured by incubation at room temperature for 30 minutes using 2.5% Triton X-100. This procedure was repeated twice, and then 50 mM Tris buffer, pH 7.8, 5 mM CaCl ₂ , 0.15 M NaCl , And 1% TritonX-100 for 24 hours. After that, the gel was stained with 0.1% Coomassie Brilliant Blue R-250. Gelatinase activity was confirmed by the presence of a clear zone as compared to a blue background. MMP1 and MMP2 were compared with each protein to determine the activity Respectively.

In addition, the present inventors Western blotted whether 20 (S) -gencenoside-Rg3 affects the expression of MMP-2 upon irradiation with ultraviolet rays. 20 (S) -gencenoside-Rg3 was transformed into HaCat Cells were pretreated and irradiated with ultraviolet light B, and the obtained cell culture was loaded on a 10% polyacrylamide gel. After electrophoresis, the proteins were transferred to a PVDF membrane, and the cells were washed with 1x TBS-Tween (Anti-MMP-2 antibody (ALX-210-753, Enzo Life Sciences, Farmingdale, NY, USA) and a secondary antibody (goat anti-rabbit IgG-pAb- HRP- 300, Enzo Life Sciences, Farmingdale, NY, USA) and visualized using enhanced West-save up ^TM (AbFrontier, Seoul, Korea).

As shown in FIG. 6, the gelatin melting activity of MMP-2 was shown to be suppressed by UV irradiation in both 20 (S) -ginnenoside Rg3 and 20 (R) -ginnenoside Rg3, whereas MMP- 1 gelatin activity. In addition, the gelatin melting activity for MMP-2 was found to be more effective to control 20 (S) -ginenoside Rg3 than 20 (R) -ginnenoside Rg3.

Furthermore, the results of the Western blot of FIG. 7 indicate that the 20 (S) -ginnenoside-Rg3 of the present invention inhibits MMP-2 protein, which is increased in expression upon UV irradiation, in a concentration-dependent manner.

Hereinafter, a formulation example of a method for producing a functional cosmetic having an ultraviolet ray blocking effect using the ginsenoside-Rg3 of the present invention is described.

< Formulation Example  1>

Flexible longevity (skin lotion)

The number of softening times was determined according to the usual method as shown in the following table.

ingredient Content (% by weight) 20 (S) -gincenoside-Rg3 or 20 (R) -gincenoside-Rg3 0.8 1,3-butylene glycol 6.0 glycerin 4.0 Oleyl alcohol 0.1 Polysorbate 20 0.5 ethanol 15.0 Benzophenone-9 0.05 Incense, preservative a very small amount Purified water to 100

< Formulation Example  2>

Nutrition lotion ( Milk lotion )

Nutrition lotion was prepared according to the usual method as shown in the following table.

ingredient Content (% by weight) 20 (S) -gincenoside-Rg3 or 20 (R) -gincenoside-Rg3 2.0 Propylene glycol 6.0 glycerin 4.0 Triethanolamine 1.2 Tocopheryl acetate 3.0 Liquid paraffin 5.0 Squalane 3.0 Macadamia nut oil 2.0 Polysorbate 60 1.5 Solitane sesquioleate 1.0 Carboxyvinyl polymer 1.0 Viechti 0.01 EDTA-2Na 0.01 Incense, preservative a very small amount Purified water to 100

< Formulation Example  3>

Nourishing cream

Nutritive creams were prepared according to conventional methods as shown in the following table.

ingredient Content (% by weight) 20 (S) -gincenoside-Rg3 or 20 (R) -gincenoside-Rg3 8.0 Cetostearyl alcohol 2.0 Glyceryl stearate 1.5 Trioctanoin 5.0 Polysorbate 60 1.2 Solitan stearate 0.5 Squalane 5.0 Liquid paraffin 3.0 Cyclone methicone 3.0 Viechti 0.05 Delta tocopherol 0.2 Concentrated glycerin 4.0 1,3-butylene glycol 2.0 Santa Sword 0.1 EDTA-2Na 0.5 Incense, preservative a very small amount Purified water to 100

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (8)

A cosmetic composition for protecting ultraviolet rays comprising ginsenoside-Rg3 as an active ingredient. The method according to claim 1,
Wherein the ginsenoside-Rg3 is 20 (S) -ginenoside-Rg3 or 20 (R) -ginnenoside-Rg3 represented by the following formula:

. The method according to claim 1,
Wherein the ginsenoside-Rg3 is contained in the composition at a concentration of 1 to 30 uM. 3. The method of claim 2,
Wherein the 20 (S) -ginsenoside-Rg3 inhibits the production of reactive oxygen species (ROS) produced by ultraviolet irradiation and has cytoprotective activity. 3. The method of claim 2,
20 (S) -gincenoside-Rg3 and 20 (R) -ginsenoside-Rg3 inhibit the activity of MMP-2 (matrix metalloproteinases-2) . The method according to claim 1,
Wherein the ultraviolet ray is UV-B having a wavelength of 280 to 320 nm. The method according to claim 1,
The composition may be in the form of a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, cleansing oil, powder foundation, emulsion foundation, wax foundation, pack, massage cream and spray &Lt; / RTI &gt; A cosmetic method characterized by having the effect of protecting the skin from ultraviolet rays by applying the cosmetic composition of claim 1 to human skin.

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