KR20180045298A - Cosmetic composition for preventing photo-aging comprising agastache rugosa extract - Google Patents

Abstract

The present invention relates to a functional cosmetic composition containing an Agastache rugosa extract, and more particularly, to a photoaging preventing or alleviating a cosmetic composition containing an Agastache rugosa extract as an active ingredient. The Agastache rugosa extract of the present invention has stable characteristics not causing side effects on skin and has an effect of inhibiting the generation of active oxygen species caused by ultraviolet irradiation in skin cells, an effect of inhibiting an activity of pro-MMP2 and pro-MMP9, and an effect of enhancing an activity of superoxide dismutase (SOD) and glutathione (GSH). Thus, the composition of the present invention containing the Agastache rugosa extract as an active ingredient can be used as a material for new functional cosmetics which can effectively inhibit skin aging caused by ultraviolet irradiation without causing irritation to the skin.

Description

[0001] The present invention relates to a cosmetic composition for preventing photo-aging comprising agar-agaric acid,

The present invention relates to a cosmetic composition for inhibiting photoaging deterioration comprising an extract of Lycopersicon esculenta as an active ingredient.

In general, when ultraviolet rays contained in sunlight are directly irradiated to the skin, it promotes the formation of erythema or melanin pigmentation in skin cells, which may be a cause of irritation and dullness. In addition, Not only does it create skin troubles by producing lipids, it can also cause skin cancer in severe cases. 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 particular, UV-B exposure increases the formation of free radicals and reactive oxygen species (ROS) in the skin and increases oxidative stress on cellular components such as DNA, cell membrane and protein It is known to induce skin aging. For this reason, a variety of studies are currently underway to prevent UV-B-induced skin damage of natural antioxidants.

Korean Patent No. 10-1194891 discloses a pharmaceutical composition for prevention and treatment of skin damage caused by ultraviolet rays, which comprises a hyaluronic acid extract. In Korean Patent No. 10-1413105, And Korean Patent No. 10-0873998 discloses a composition for external use for sun protection against sunscreen containing an extract of Succinic acid as an active ingredient.

On the other hand, Agastache rugosa is a perennial herb that grows in mountains and branches of East Asia including Korea and Japan, and its growth environment is abundant in the subtillies of the soil and grows in sunny or semi-silky. Its height is 40 ~ 100㎝. Leaves are opposite, 5 ~ 10㎝ in length, 3 ~ 7㎝ in butterfly, pointed end and heart shaped. Flowers bloom in purple, 5 ~ 15㎝ in length, 2㎝ in butterfly, and run in the shape of an umbrella at the ends of the branches and main stem. The fruit ripens in October ~ November, and the ovary has turned into a dark brown color and contains many seeds in minute form. It is mainly used for ornamental purposes.

The research on this breeding has not yet been carried out so far, and it is known that there is a current anti-inflammatory and anti-arteriosclerotic activity, a nerve inflammation prevention effect, a cholera treatment effect, an antibacterial activity and an antiviral activity, There is no research on the skin cell protection effect and the photoaging inhibition function attributable thereto.

Korean Patent No. 10-1194891 Korean Patent No. 10-1413105 Korean Patent No. 10-0873998

Accordingly, the present inventors have found that the extract of Bombyx mori L. can suppress the production of active oxygen induced by ultraviolet rays, proMMP-2 and proMMP-9, and at the same time, the amount of superoxide dismutase (SOD) and glutathione The present invention has been accomplished by confirming that it is possible to prevent and improve photoaging through inhibition of skin cell damage upon ultraviolet irradiation.

Accordingly, it is an object of the present invention to provide a cosmetic composition for preventing or improving photoaging, which comprises an extract of Rice bran extract as an active ingredient.

Another object of the present invention is to provide a cosmetic method which is characterized in that the cosmetic composition of the present invention is applied to human skin and has an effect of preventing skin aging by ultraviolet rays.

In order to achieve the object of the present invention, the present invention provides a cosmetic composition for preventing or improving photoaging, which comprises an extract of Rice bran extract as an active ingredient.

In one embodiment of the present invention, the extract of Bombyx mori is a hot-water extract of Bombyx mori.

In one embodiment of the present invention, the extract of Bombyx mori extract may inhibit skin cell damage by ultraviolet rays; Inhibition of active oxygen species production; inhibiting the activity of pro-MMP2 and pro-MMP9; Or superoxide dismutase (SOD) and glutathione (GSH) activity.

In one embodiment of the present invention, the extract may be contained at a concentration of 10 to 200 ug / ml based on the total weight of the composition.

In an embodiment of the present invention, the ultraviolet ray may be ultraviolet ray B.

In one embodiment of the present invention, the composition may be a skin lotion, a skin softener, a skin toner, an astringent, a lotion, a milk lotion, a moisturizing lotion, a nutrition lotion, a massage cream, a nutritive cream, And may be formulated into a product selected from the group consisting of a pack, a soap, a shampoo, a cleansing foam, a cleansing lotion, a cleansing cream, a body lotion, a body cleanser, a latex, a lipstick, a makeup base, a foundation, a press powder and a loose powder have.

The present invention also provides a makeup method characterized in that the cosmetic composition of the present invention is applied to human skin and has an effect of preventing skin aging by ultraviolet rays.

The present invention relates to a functional cosmetic composition containing a pine needle extract, and more particularly, to a cosmetic composition for prevention or improvement of photoaging comprising a pine needle extract as an active ingredient. The extract of the present invention has a stable characteristic that it does not cause adverse effects on the skin and can effectively inhibit the production of reactive oxygen species to skin cells upon irradiation with ultraviolet rays and has the effect of inhibiting the activity of pro-MMP2 and pro-MMP9 The composition of the present invention comprising SOD (superoxide dismutase) and GSH (glutathione) as an active ingredient is effective as a new functional cosmetic composition capable of effectively inhibiting skin aging caused by ultraviolet irradiation without stimulating the skin Can be used.

FIG. 1 shows the results of analysis of ABTS ⁺ radical scavenging ability of the extract of hot water extract according to the present invention (ARE: hot water extract of the present invention, AA: ascorbic acid)
FIG. 2 is a graph showing the inhibitory effect of UV-B irradiation on the HaCaT keratinocytes cell line according to the treatment concentration of hot water extract of Baekryeo extract. 2a is the result of DCFH-DA measurement, 2b is dihydrorhodamine 123, and 2c shows the result of measurement of 2-hydroxyethidium (2-HE).
FIG. 3 shows the results of MTT analysis of the HaCaT keratinocytes cell line for cytotoxicity according to the treatment concentration of hot water extract of the present invention under UV-B irradiation.
Fig. 4 shows the results of the echinocarcinoma analysis of the effect of the hot water extract on the growth of proMMP-2 and proMMP-9 increased by UV-B irradiation.
FIG. 5 shows the result of western blotting to confirm whether the levels of proMMP-2 and proMMP-9 protein increased by UV-B irradiation can be reduced by the hot water extract of Rice.
FIG. 6 shows the results of analysis of whether the activity of total GSH and SOD reduced by UV-B irradiation can be increased by the hot water extract of the present invention.

The inventors of the present invention confirmed that the extract of Lycopersicon esculentum has such a function while studying a new cosmetic material that can prevent and inhibit skin photoaging due to its excellent skin protecting effect against ultraviolet rays without irritating the skin.

Accordingly, the present invention is characterized by providing a cosmetic composition for preventing or improving photoaging comprising an extract of Lycopersicon esculenta as an active ingredient.

Particularly, the present inventors searched for a material that is derived from a natural substance and stable to the human body and capable of exhibiting excellent skin photo-aging effect, and focused on the extract of Bombyx mori. The extract of Bombyx mori, And MMP inhibitory activity.

Specifically, according to one embodiment of the present invention, it was confirmed that the extract of Bombyx mandarin hydrothermal extract has an active oxygen species inhibitory function and the ABTS + radical scavenging ability analysis shows that the Bombyx mori extract has a high ABTS + radical scavenging ability (see FIG. 1 ).

Skin aging, on the other hand, can be classified into two categories: internal aging and external aging. Intrinsic aging is a process in which the physiological function of the body is lowered with age, and aging extrinsic aging is an aging phenomenon caused by external factors such as ultraviolet (UV), dry air, reactive oxygen species and stress. These factors lead to skin aging. When skin aging occurs, the skin becomes keratinized, the fusion rate of epidermal cells gradually decreases, the epidermal layer becomes thinner, the stratum corneum becomes thicker, and the boundary line with the dermal layer becomes straight. In the dermis, the aging of the human skin fibroblast deteriorates the productivity of the fibrous material and the substrate, increases the peroxide level in the cell rapidly, is affected by various contaminants and stresses, The metabolism activity of the cells is lowered, resulting in wrinkles due to functional and structural damage, thereby promoting skin 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.

In this respect, it has been confirmed through experiments that the present invention can effectively inhibit free radical generation in skin cells due to ultraviolet irradiation and inhibit the production of reactive oxygen species.

In addition, in order to confirm the stability of the extract of Bombyx mori L. on the skin cells, the extract of Bombyx mori L. extract was treated at different concentrations and the degree of apoptosis was measured by MTT analysis. As a result, (See Fig. 3).

Therefore, the extract of the present invention does not exhibit toxicity to skin cells, and therefore, it can be safely used for the production of cosmetics applied to the skin.

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. It is known that MMPs are activated by protein hydrolysis after being synthesized as an inactive precursor, and that the activity of MMPs is regulated by gene expression, activation of inactive enzymes, and inhibition of enzyme activity. 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.

These MMPs are known to increase expression of reduced collagen synthesis through complex signal transduction pathways in skin exposed to ultraviolet light (Lee et al., J. Dermatol Sci, 17, 182, 1998) A is known to activate MMP-1, and it is known that the activity of MMP-2 and MMP-9 is increased upon UVB irradiation. It has been reported that increasing the activity of MMP-2 and MMP-9 in the skin caused by ultraviolet irradiation significantly affects the collagen collapse of the dermal layer by collapsing collagen in the skin and plays a very important role in photoaging. 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.

Therefore, in order to confirm whether the extract of the present invention can inhibit the activity and the protein level of MMP-2 and MMP-9 which are activated upon irradiation with ultraviolet rays, The activity of pro-MMP2 and pro-MMP9 was analyzed by mammography and the level of protein was Western blot.

As a result, activity of pro-MMP2 and pro-MMP9 activated by ultraviolet B irradiation was inhibited in the group treated with the extract of Bombyx mori, and the protein level was also decreased. (Fig. 4 and Fig. 5).

In addition, it was confirmed that the extract of Rice bark extract of the present invention has an activity to re-enhance activity of SOD (superoxide dismutase) and glutathione (GSH) reduced by ultraviolet irradiation.

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

The extract of the present invention may be obtained by extracting and isolating from nature using an extraction and separation method known in the art, and the 'extract' defined in the present invention may be prepared by extracting And includes, for example, a crude extract of a syrup, a polar solvent-soluble extract or a non-polar solvent-soluble extract. Preferably, it may be an extract obtained from the leaves of the foxtail.

Examples of a suitable solvent for extracting the extract from the buckwheat may include water or an organic solvent. Examples of the solvent include, but are not limited to, purified water, methanol, ethanol, propanol, isopropanol isopropyl alcohol, isopropanol, butanol and the like, an alcohol having 1 to 4 carbon atoms, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride ), Hexane, and cyclohexane, may be used alone or in combination. Preferably, water can be used.

As the extraction method, any one of the methods such as hot water extraction, cold extraction, reflux cooling, solvent extraction, steam distillation, ultrasonic extraction, elution, and compression can be used, and hot water extraction can be preferably used .

In addition, the desired extract may be further subjected to a conventional fractionation process or may be purified using a conventional purification method.

The syrup extract may be contained at a concentration of 10 to 200 ug / ml based on the total weight of the composition of the present invention.

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.

Furthermore, the components contained in the cosmetic composition of the present invention may contain, as an active ingredient, components commonly used in cosmetic compositions in addition to the extract of the leaf extracts, and may contain conventional components such as antioxidants, stabilizers, solubilizers, vitamins, Supplements, 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, methyl taurate, 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 extract of Rice bran extract of the present invention as an active ingredient. That is, the present invention provides a cosmetic method characterized by having a cosmetic composition of the present invention applied to human skin and having an effect of preventing skin aging by ultraviolet rays.

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. As a specific example, the soap is liquid soap, powdered soap, solid soap and oil soap, and the surfactant-containing cleansing formulation is a cleansing foam, a cleansing water, a cleansing towel and a cleansing pack, , 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. These examples are for further illustrating the present invention, and the scope of the present invention is not limited to these examples.

< Example  1>

Reagents and sample preparation

Diethyleneglycol, naringin, ABTS (2,2-azino-bis 3-ethylbenzthiazoline-6-sulfonic acid), AA (ammonium persulfate, ascorbic acid), MTT (3- (2 ', 7'-dichlorodihydrofluorescein diacetate), DHR-123 (dihydrorhodamine 123), DHE (dihydroethidium), DTNB (5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) , 5'-dithiobis (2-nitrobenzoic acid), glutathione reductase (GR), catalase, xanthine, xanthine oxidase and NADPH were purchased from Sigma Aldrich Respectively. The cell lysis buffer (25 mM Tris-phosphate (pH 7.8), 2 mM diaminocyclohexane- N , N , Nv , Nv- tetraacetic acid, 2 mM dithiothreitol, 10% glycerol, 1% Triton X- Were purchased from Mega Corporation and used.

The plant was purchased from Chuncheon Mart in Gangwon Province and the identification of the plant was confirmed by Chungcheon University professor of organic science. The voucher specimen of the present invention was stored in a plant sample room of Kangwon National University and received the approval number KWNU90446.

Statistical processing

Experimental results were expressed as mean ± SD. Unpaired Student's t-test was performed to analyze the results. A p-value <0.05 was considered statistically significant. IC50 values were calculated from dose / response linear regression plots.

< Example  2>

Berry leaf Heat number  Extract preparation

The leaves of the dried bamboo shoots were finely crushed in liquid nitrogen, and distilled water corresponding to 10 times the weight of the bamboo leaf was added and mixed. Then, the mixture was subjected to hot water extraction at 90 ° C for 4 hours under reflux in a water bath. When the temperature of the hot-water extract was lowered, it was filtered using a filter paper, and dried using a freeze dryer to obtain a hot-water extract (called an ARE) The yield was 10.4%. For the subsequent experiments, the hot water extract of Baekjang was obtained by dissolving in dimethyl sulfoxide.

< Example  3>

Total flavonoid content and antioxidant activity measurement

The analysis of total flavonoid content in the hot-water extract of the bladder leaves obtained in Example 2 was carried out as described in Prev. Nutr. Food Sci. 17 (2012) 122-128, that is, 0.5 ml of diethylene glycol and 0.05 ml of ARE at different concentrations were mixed with 0.05 ml of 1 N NaOH and stirred for 1 hour And reacted at 37 ° C. The absorbance at 420 nm was measured using a microplate reader, in which Naringin was used as a standard marker for the determination of total flavonoid content (ranging from 0 to 1 mg / ml). The total flavonoid content was expressed in mg / g ARE (mg / g NE).

The antioxidant activity was analyzed by ABTS ⁺ radical scavenging assay. After 0.12 mM ammonium persulfate was added to the ABTS storage solution (0.07 mM) and mixed, it was stored in a dark room for 16 hours before use for analysis . Then, 0.01 ml of ARE (the fresh water extract of the present invention) was added to 0.29 ml ABTS ⁺ solution for each concentration, and the volume was adjusted to 1 ml with ethanol. The reaction solution was reacted in the dark room for 15 minutes. At this time, ascorbic acid (AA) was used as a positive control. Absorbance was measured at 745 nm, and the degree of inhibition (%) by ARE was calculated by the following equation. The analytical results show ABTS ⁺ radical 50% scavenging activity (SC ₅₀ ) by ARE or AA.

Inhibition (%) = [(Control - Test) / Control] x100

As a result of the analysis, the total flavonoid content of the dried hot water extract obtained by the method of the present invention was found to be 22.8 ± 7.6 mg naringin equivalent / g ARE based on the dried amount, and the antioxidant ability of the hot water extract of Bombyx mori, that is, SC ₅₀ was 836.9 μg / mL, and the SC ₅₀ of ascorbic acid used as a positive control was 59.7 μg / mL (see FIG. 1). From these results, the inventors of the present invention have found that the hot water extract of the present invention contains a large amount of flavonoids and has relatively excellent antioxidant ability.

< Example  4>

Fried Heat number  Production of active oxygen by ultraviolet rays of extract Inhibition  analysis

The HaCaT keratinocyte cell line was subjected to the following experiment in order to analyze the activity of inhibiting the active oxygen generated by ultraviolet rays from the hot water extract of the leaf of the present invention.

First, the HaCaT keratinocyte cell line, a keratinocyte cell line, was purchased from ATCC. Using DMEM medium containing 10% heat-inactivated FBS, 100 units / ml penicillin and 100 ug / ml streptomycin, 5% &Lt; / RTI &gt; The HaCaT keratinocyte cell line cultured in the above manner was treated with hot water extracts (0, 10, 50, and 200 ug / ml) for 30 minutes and then irradiated with ultraviolet B, UV light (peak, 312 nm; model VL-6M) using a radiometer (model VLX-3W, Vilber Lourmat, Marine, France) equipped with a model CX-312, Vilber Lourmat, , Vilber Lourmat, Marine, France).

After irradiation with ultraviolet light B for 18 hours, the cells were washed twice with PBS, left in ice for 5 minutes, and scraped with a cell scrapper. The cells were centrifuged at 15000 rpm for 10 minutes, and the cell pellet was dissolved in a cell lysis buffer and allowed to stand in ice again for 30 minutes. The content of protein in the cell lysate was measured by Bradford reagent and BSA (bovine serum albumin) was used as a standard protein.

Measurement of active oxygen generated in the cell is active oxygen species (ROS) in reaction fluorescence samples were carried out using DCFH-DA, DCFH-DA is reacted with ROS DCF (fluorescent 2 ', 7'-dichlorofluorescein; λ excitation = 485 nm, and [lambda] _emission = 530 nm) to induce a continuous oxidation reaction. DHR-123 and DHE were used as the other two ROS fluorescent probes. When reacted with active oxygen species, they were 123 (RH-123; λ _excitation = 500 nm, λ _emission = 535 nm) and 2-hydroxyethidium (DHE); generates a _{λ excitation = 480nm, λ emission =} 525nm). Each of the cell lines was treated with the extract of the present invention, ARE, 20 μM DCFH-DA, 5 μM DHR-123, or 5 μM DHE for 30 minutes at 37 ° C., followed by washing twice with 1 mL of FBS-free medium. Cells were lysed with 1 mL of FBS-free medium and irradiated with 70 mJ / cm ² UV-B. Immediately after irradiation with ultraviolet light, the amount of reactive oxygen species generated in the cells was monitored by using a Multi-Mode Microplate Reader (Synergy ^TM Mx, BioTekInstruments, Winooki, VT, USA).

As a result of the analysis, as shown in FIG. 2, in the result of DCF fluorescence analysis, in the group irradiated only with ultraviolet rays, 9.6 times more active oxygen species were generated than in the group not irradiated with ultraviolet rays, and the hot water extract In one group, the production of reactive oxygen species was inhibited in a concentration-dependent manner (see Fig. 2a). Specifically, when the extract was treated at the concentrations of 10, 50 and 200 ug / ml, the production of reactive oxygen species decreased to 83.1%, 55.8% and 33.8%, respectively.

DHR-123, which was used as a ROS dye, showed 3.8-fold increase in active oxygen species in UV irradiation, whereas active oxygen species in the group treated with 10, 50 and 200 ug / ml of hot- (DHE) fluorescence spectra were also reduced to 78.9%, 40.8% and 21.1%, respectively (see FIG. 2b). The results of the 2-HE (DHE) (See FIG. 2C).

As described above, in all of the three reagents which can confirm the degree of production of reactive oxygen species, it was found that the extract of Rice bran extract had a concentration-dependent inhibitory effect on the production of reactive oxygen species, and that the production of reactive oxygen species was inhibited by 50% Looking at the IC50 values, DCFH-DA, DHR-123 and DHE were found to be 110.5 ug / ml, 69.3 ug / ml and 45.1 ug / ml, respectively.

< Example  5>

Fried Heat number  Analysis of cytotoxicity of extracts

MTT analysis was performed to determine whether the extract of the present invention was toxic to cells. The MTT reaction is a method of analyzing the amount of formazan generated in the reduction reaction of MTT in the mitochondria of living cells by measuring the absorbance at 540 nm.

The present inventors performed MTT analysis on the HaCaT keratinocyte cell line treated with the hot-water extract of L. orientalis leaf under ultraviolet irradiation condition in Example 4 above.

As a result of the analysis, as shown in Fig. 3, there was no change in the cell survival rate regardless of the presence or absence of ultraviolet irradiation and whether or not the present invention was treated with the hot water extract of the present invention. Therefore, it can be seen from the above results that the hot water extract of the present invention is a safe extract that does not cause toxicity to cells.

< Example  6>

Fried Of hot-water extract  pro- MMP2  And pro- Of MMP9  Inhibitory activity assay

<6-1> Zymography  analysis

The present inventors analyzed gelatinolytic activity of pro-MMP2 and pro-MMP9, which are precursors thereof, by gel permeation analysis, in order to analyze the effect of the extract on the MMP2 and MMP9.

The HaCaT keratinocyte cell line used in the above example was treated with the hot-water extract of the present invention for each concentration and washed with PBS, and the culture medium of each test group was collected for proliferation of pro- -MMP2 and pro-MMP9 gelatin solubility. The prepared cell lysate samples were electrophoresed on non-reducing conditions on a gel made by adding 1 mg / ml of gelatin to a standard laemmli acrylamide polymerization mixture (8%). After the electrophoresis, the detergent solution (2.5% Triton X-100) was treated for 15 minutes twice to remove the SDS remaining on the gel. The gel was then reacted with substrate buffer (50 mM Tris-HCl, 5 mM CaCl 2, NaN 3) for 24 h at 37 ° C. The activity of pro-MMP2 and pro-MMP9 was measured by 0.1% Coomassie Brilliant Blue R-25 solution. The extent of activity was determined by measuring the degree of gelatin dissolution by the band intensity. In addition, the active bands of pro-MMP2 and pro-MMP9 can be identified based on their molecular weights, which are 72 kDa and 92 kDa, respectively.

As shown in Table 4, gelatin solubility of pro-MMP2 and pro-MMP9 were increased 5.3 and 3.6 times, respectively, compared with the group not irradiated with ultraviolet light, The pro-MMP2 and pro-MMP9 activities were reduced in a concentration-dependent manner in the group treated with the hot-water extract of L. vannamei. When the extract treatment concentration was 10, 50, 200 ug / ml, (Fig. 4A), and the activity of pro-MMP9 decreased to 72.0, 44.0 and 24.0%, respectively (Fig. 4B).

The IC50 of the hot-water extract of Baekjiae leaf, the degree of decrease of pro-MMP9 activity by ultraviolet B irradiation, was 76.6 ug / ml. FIG. 4C is a photograph of gels stained with Coomassie Brilliant Blue R-250 by loading the same amount of the used culture medium. It is known that the extract of the present invention has a concentration-dependent effect on both pro-MMP2 and pro-MMP9 I could.

Therefore, the inventors of the present invention have found that the hot water extract of Bombyx mori extract has an activity of significantly inhibiting the activities of pro-MMP2 and pro-MMP9 by ultraviolet irradiation.

<6-2> Western Blot  analysis

Western blotting was performed as follows to determine whether the extract of Bombyx mori L. influenced pro-MMP2 and pro-MMP9 protein levels as well. Cell lysates prepared in the above Example <6-1> were prepared, electrophoresed by 10% (w / v) SDS-PAGE, proteins were transferred with PVDF, Were incubated with anti-MMP-2 (ALX-210-753, Enzo Life Sciences, Farmingdale, NY, USA) and anti-MMP-9 (3852S, Cell Signaling Technology, Danvers, Lt; / RTI &gt; After the second antibody; After reacting for 1 hour at room temperature (goat anti-rabbit IgG-pAb -HRP-conjugate ADI-SAB-300, Enzo Life Sciences, Farmingdale, NY, USA), enhanced West-save up TM ( AbFrontier, Seoul, Korea) to analyze the level of the target protein.

As a result of the analysis, as shown in FIG. 5, protein level of pro-MMP2 was increased about 5.8 times as compared with the group not irradiated with ultraviolet ray B at ultraviolet B irradiation, , Pro-MMP2 protein levels were reduced to 84.4, 75.0 and 35.9%, respectively, depending on the treatment concentration (10, 50, 200 ug / ml) (see FIG. In addition, pro-MMP9 also showed an increase in protein level by about 6 times when irradiated with ultraviolet B, but in the group treated with the extract of the present invention, pro-MMP9 protein level was dependent on treatment concentration (10, 50, 200 ug / ml) Respectively, to 95.5, 72.7 and 50.0%, respectively (see FIG. 5B). The IC50 values for the inhibition of pro-MMP2 and pro-MMP9 activity by ultraviolet B irradiation were 147.6 ug / ml and 189.7 ug / ml, respectively.

Based on these results, it can be seen that the hot water extract of the present invention has the action of simultaneously reducing and inhibiting the activities and protein levels of pro-MMP2 and pro-MMP9 induced by ultraviolet B irradiation.

< Example  7>

Fried Of hot-water extract GSH  And SOD activity acceleration assay

<7-1> By ultraviolet B irradiation Reduced GSH  lift Control ability  analysis

GSH is a nonprotein antioxidant that plays an important role in protecting against oxidative stress and improving oxidation - reduction. In order to examine whether the present invention's hydrothermal extracts of the present invention, which confirmed the action of active oxygen species and pro-MMP2 and pro-MMP9, were also involved in GSH, the content of total GSH in HaCaT keratinocyte cells under ultraviolet B irradiation The effect of the extract was analyzed. For this purpose, GR-based enzymatic regeneration assay was performed on the cell lysates used in the usual examples. To this end, a reaction mixture (200 ul) containing 175 mM KH ₂ PO ₄ , 6.3 mM EDTA, 0.21 mM NADPH, 0.6 mM DTNB, 0.5 units / mL GR and cell lysate was reacted at 25 ° C, To measure the absorbance. The total GSH content was measured by using a calibration curve using various concentrations of GSH as a standard. The total amount of protein contained in the cell lysate was normalized, and the total amount of GSH was expressed in ug / ml.

As a result, as shown in FIG. 6A, the total amount of GSH was increased depending on the treatment concentration of the extract of the present invention. When the extract was treated at a concentration of 10, 50 and 200 ug / ml, And 1.3 times, 1.6 times, and 2.1 times, respectively, compared with the untreated control group.

<7-2> By ultraviolet B irradiation Reduced  Upside of SOD Control ability  analysis

SOD with catalase and glutathione peroxidase function are the major antioxidant enzymes. Therefore, the inventors of the present invention confirmed the effect of the extract of the present invention on SOD activity. For this purpose, reduction of cytochrome c was performed using the xanthine / xanthine oxidase system in the cell lysate used in Example <7-1> Respectively. Specifically, 200 μl of a reaction mixture containing 50 mM phosphate buffer (pH 7.4), 0.01 units / mL xanthine oxidase, 0.1 mM EDTA, 1 μM catalase, 0.05 mM xanthine, 20 μM cytochrome c and cell lysate was prepared, nm was measured. The SOD activity was normalized to the protein content of the cell lysate and expressed as _? A ₅₅₀ / min / mg protein.

As shown in FIG. 6B, ultraviolet B irradiation reduced total SOD activity (Cu, Zn-SOD plus Mn-SOD), but SOD activity was increased when the present extract of the present invention was treated , And SOD activity was increased 2.6, 4.2, and 4.7 times, respectively, compared to the control without the extract of the present invention when the extract treatment concentrations were 10, 50 and 200 μg / mL. Therefore, it can be seen that the hot water extract of the present invention has an action to improve the activity of SOD reduced by ultraviolet B irradiation.

In conclusion, based on the above results, it can be seen that the hot water extract of the present invention has excellent activity of increasing reduced SOD and GSH activity and protein level in skin cells due to irradiation with ultraviolet B, Inhibitory activity and the activity of pro-MMP2 and pro-MMP9, and ultimately, it can be effectively used for the manufacture of cosmetics for prevention and improvement of skin damage due to photoaging.

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 (7)

A cosmetic composition for prevention or improvement of photoaging comprising an extract of Lycopersicon esculenta as an active ingredient. The method according to claim 1,
The cosmetic composition for prevention or improvement of photoaging according to any one of claims 1 to 3, wherein the extract is a hot-water extract of leaf blossom. The method according to claim 1,
The bamboo shoot extract inhibits skin cell damage by ultraviolet rays; Inhibition of active oxygen species production; inhibiting the activity of pro-MMP2 and pro-MMP9; Or superoxide dismutase (SOD) and glutathione (GSH) activity of the cosmetic composition. The method according to claim 1,
The cosmetic composition for prevention or improvement of photoaging according to any one of claims 1 to 3, wherein the extract is contained at a concentration of 10 to 200 ug / ml based on the total weight of the composition. The method of claim 3,
Wherein the ultraviolet ray is ultraviolet ray B. 2. The cosmetic composition for preventing or improving photoaging according to claim 1, The method according to claim 1,
The composition may be in the form of a skin lotion, a skin softener, a skin toner, an astringent, a lotion, a milk lotion, a moisturizing lotion, a nutrition lotion, a massage cream, a nutrition cream, a moisturizing cream, a hand cream, A cosmetic composition for prevention or improvement of photoaging according to the present invention is characterized in that it is formulated as one kind selected from the group consisting of cleansing lotion, cleansing cream, body lotion, body cleanser, emulsion, lipstick, makeup base, foundation, press powder and loose powder . The cosmetic composition according to claim 1, which is applied to human skin and has an anti-aging effect on skin caused by ultraviolet rays.

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