KR20030004889A - Cosmetic composition containing a D-fructose 1,6-diphosphate inhibiting a skin cell injury by the ultraviolet - Google Patents

Abstract

PURPOSE: A cosmetic composition containing D-fructose 1,6-diphosphate and a salt thereof is provided which causes no skin irritation and inhibits skin damage caused by irritation such as UV ray. Therefore it can be used as a cosmetic material playing a role of endogenous antioxidant substances for improving self-reducing power of skin cells. CONSTITUTION: The cosmetic composition contains 0.0001 to 20 parts by weight of D-fructose 1,6-diphosphate and a salt thereof, based on the total weight of the composition and reduces the concentration of an inflammatory intermediate factor of prostaglandins by inhibition of activity of cyclooxygenase-2.

Description

Cosmetic composition containing fructose 1,6-diphosphate and salts thereof to inhibit skin cell damage caused by ultraviolet light {1,6-diphosphate inhibiting a skin cell injury by the ultraviolet}

The present invention relates to a cosmetic composition containing fructose 1,6-diphosphate and salts thereof that inhibit skin cell damage caused by ultraviolet light.

Cells cause cell damage response by inducing various biochemical changes in cells by stimulating cell damage such as ultraviolet rays, environmental pollutants, and sudden changes in temperature. It is also known that skin aging during cell damage responses is due in part to changes in the physiological function of the skin resulting from oxidative damage responses induced by these external stimuli.

In other words, if cell membranes of skin cells are continuously damaged by free radicals or other radicals, cross-linkage of proteins or lipoproteins is increased or degraded, thereby increasing the amount of lipid peroxide. Thus, the permeability of the cell membrane changes to disrupt the ion balance in the cell, and this change prevents the release of metabolic debris in the cell and also reduces the cell's ability to detoxify the metabolic debris. Increasing the concentration of potassium in the cell increases the colloid density, which interferes with the synthesis of mRNA and protein, resulting in a lower ability to heal cell damage.

As the cellular level changes persist, the tissue structure loses regularity, while at the same time the individual cells grow relatively, while the total cell number decreases. In addition, local scars appear on the dermis and the thickness of the dermis decreases due to the reduction of collagen fibers or elastic fibers. Crosslinking between collagen fibers also increases and the fine structure of the elastic fibers is also damaged, resulting in a decrease in the elasticity of the skin as a whole. That is, oxidative damage of skin cells by external stimuli ultimately promotes aging of the skin.

Substances that cause such oxidative cell damage include hydrogen peroxide (H ₂ O ₂ ), superoxide anion, and hydroxy radicals caused by oxidative stress. To eliminate endotoxins, our bodies are equipped with endogenous antioxidant systems such as glutathione and superoxide dismutase (SOD). However, excessive increase in intracellular metabolism or exposure of the body to exogenous stress, such as infection, excessive exercise, radiation or compounds, may exceed these endogenous antioxidant capacities, resulting in free radical cell damage. . Therefore, a substance that increases the endogenous antioxidant power of the skin cell itself, that is, increases the physiological reducing power of the cell, is capable of suppressing cell damage from stimulation such as ultraviolet rays. Therefore, conventionally, antioxidants such as vitamin C, vitamin E, and green tea catechin, which have excellent radical scavenging effects, are commonly used in cosmetics to suppress such cellular damage. However, the conventional method, which relies on physicochemical radical scavenging, has a problem of chemical stability, such as its effect is rapidly decreased when applied to a product. Can cause skin cell irritation.

In order to solve the above problems, the present inventors randomly select hundreds of compounds to find an antioxidant which is a cell damage protecting agent without relying on physicochemical radical scavenging to study the function of inhibiting skin cell damage by ultraviolet rays. As a result, it was found that fructose 1,6-diphosphate has an excellent inhibitory effect on skin cell damage.

Furthermore, as a result of the extensive research by the present inventors, when fructose 1,6-diphosphate and salts thereof are contained in an amount of 0.0001 to 20% by weight based on the total weight of the composition, it inhibits cell damage and provides excellent skin stability. It was found that it can be represented.

Accordingly, it is an object of the present invention to find a substance that inhibits skin cell damage, to suppress skin cell damage continuously exposed to oxidative stress, and to provide a cosmetic composition having excellent skin stability.

1 is a graph showing the effect of inhibiting keratinocyte damage by fructose 1,6-diphosphate ultraviolet (UV B 30mJ / ㎠).

2 is a graph showing the effect of inhibiting the active oxide (ROS) production of keratinocytes by ultraviolet rays of fructose 1,6-diphosphate.

3 is a graph showing the change in glutathione (GSH) amount of keratinocytes after UV irradiation.

4 is a graph showing the effect of inhibiting prospalandin biosynthesis of keratinocytes by ultraviolet rays of fructose 1,6-diphosphate.

In order to achieve the above object, the cosmetic composition of the cell damage inhibiting function according to the present invention is characterized in that it contains fructose 1,6-diphosphate and salts thereof in an amount of 0.0001 to 20% by weight based on the total weight of the composition do.

Hereinafter, the present invention will be described in more detail.

The composition for inhibiting skin cell damage of the present invention contains fructose 1,6-diphosphate and salts thereof as an active ingredient. That is, fructose 1,6-diphosphate may provide a cosmetic composition having excellent skin safety while having a glutathion production promoting effect and a cyclooxygenase-2 enzyme inhibitory effect.

In addition, specific examples of the fructose 1,6-diphosphate may also be used in the form of salts include sodium salts and potassium salts.

The fructose 1,6-diphosphate of the present invention preferably contains an amount of 0.0001 to 20% by weight based on the total weight of the composition, and the sodium and potassium salts of fructose 1,6-diphosphate are also 0.0001 to 20% by weight. It is preferable to contain as.

Cosmetic compositions containing fructose 1,6-diphosphate of the present invention and salts thereof may be formulated into common formulations that can be applied to the skin, for example, lotions, creams, lotions, skin lotions, packs, foundations It may be formulated as a liquid soap, a solid soap, a human cleanser such as a cleansing foam as well as a cosmetic such as the like. Each of these formulations may contain various bases and additives as appropriate depending on the formulation of the formulation, and the types and amounts of these components can be easily selected by the inventors.

Hereinafter, the present invention will be described in detail with reference to Examples and Test Examples, but the present invention is not limited only to these examples.

In this example, the skin cells were damaged by ultraviolet rays, and the fructose 1,6-diphosphate used in this example was Sigma (St. Louis, MO). , USA) was used.

Test Example 1 Effect of Fructose 1,6-diphosphate on Inhibition of Cell Damage by Ultraviolet Rays

The keratinocytes (keratinocyte) isolated from the skin tissue of the person in each well of a 96 well (well) type cell culture plate 1 × 10 ⁴ by one insert was attached for 24 hours. After 18 hours, the culture was removed and 50 μl of phosphate buffered saline (PBS) was added to each well. The keratinocytes were irradiated with ultraviolet light 30mJ / cm 2 using an ultraviolet B lamp (Model: F15T8, UV B 15W, Sankyo Dennki, Japan), and then PBS was removed and each cell was cultured with keratinocyte culture medium ( keratinocyte growth media, Clonetics, BioWhittacker, MD, USA) 200 μl was added.

Fructose 1,6-diphosphate was treated here at concentrations of 1 mM, 5 mM, 10 mM and 20 mM, and then incubated for 16-20 hours. After a certain time, an appropriate amount of the culture supernatant was taken to quantify the amount of lactate dehydrogenase (LDH), an indicator of cell damage. The amount of LDH in the culture supernatant was measured using Cytotox 96 non-radioactive cytotoxicity assay kit of Promega (Madison, WI, USA), the results are shown in Figure 1 below.

As shown in Figure 1, fructose 1,6-diphosphate inhibited the increase of LDH due to human keratinocyte damage by ultraviolet light by about 86.3% at 20mM, about 64.7% at 10mM. In the above, it was confirmed that fructose 1,6-diphosphate has an effect of protecting skin cells against external stimuli such as ultraviolet rays.

Test Example 2 Inhibition Effect of Fructose 1,6-diphosphate Formation of Reactive Oxygen Species

In order to suppress the production of free radicals that are increased in skin cells by ultraviolet rays, 5 × 10 ⁴ keratinocytes isolated from human epidermal tissue were placed in each well of a 24-well cell culture plate. Attached for hours. After 16 hours, 10 mM of fructose 1,6-diphosphate was treated, and after 2 hours, the culture medium was removed, and 100 μl of phosphate buffer solution (PBS) was added to each house. The keratinocytes were irradiated with UV 30mJ / cm 2 using an ultraviolet B lamp (Model: F15T8, UV B 15W, Sankyo Dennki, Japan), and then PBS was removed and each cell was prepared with keratinocyte culture medium 200. Μl was added. Fructose 1,6-diphosphate was again treated and the amount of reactive oxygen species (ROS) increased by UV stimulation at certain time points was quantified. The amount of ROS was quantified with reference to Tan's method for measuring the fluorescence of dichlorofluorescin diacetate (DCF-DA) oxidized by ROS (Tan et al., 1998, J. Cell Biol. Vol. 141). , pp1423-1432), and the results are shown in FIG. 2.

In Figure 2 it was found that fructose 1,6-diphosphate inhibits the production of reactive active oxide (ROS) known to cause skin cell damage by ultraviolet light.

Test Example 3 Fructose 1,6-diphosphate Glutathione (GSH) production promoting effect

In order to test the effect of promoting the production of glutathione (GSH) as an indicator of reducing power in the cells, human keratinocytes (keratinocytes) were put in 5 × 10 ⁵ round cell culture plates 10 cm in diameter and attached for 24 hours. After 18 hours, the culture was removed and 2 ml of phosphate buffered saline (PBS) was added to each well. The keratinocytes were irradiated with UV light 30mJ / cm2 using an ultraviolet B lamp (Model: F15T8, UV B 15W, Sankyo Dennki, Japan), and then PBS was removed and each cell was subjected to keratinocyte culture. 5 ml was added. Fructose 1,6-diphosphate was again treated and the amount of GSH changed by UV stimulation at certain time points was quantified. GSH quantification was performed according to Akerboom and Sies's method of measuring the absorbance of 5-thio-2-nitrobenzoate produced by reduction of GSH by 5,5-dithiobis-2-nitrobenzoic acid (Akerboom & Sies, 1981). , Methods Enzymol.Vol 77, pp 373-382), and the results are shown in FIG. 3.

As shown in Figure 3, it was confirmed that the amount of GSH of keratinocytes of the skin is increased by fructose 1,6-diphosphate. Since GSH has a high reducing power in skin cells, it is possible to reduce the amount of ROS generated when cell damage caused by UV rays is induced, and from the results of FIGS. 2 and 3, fructose 1,6-diphosphate is used to oxidize skin cells. It can be seen that it is a substance that inhibits damage.

Test Example 4 Inhibitory Effect of Fructose 1,6-diphosphate on Prostaglandin E2 Biosynthesis by Inhibiting Cyclooxygenase-2 Activity

It has been reported that skin cells express large amounts of cyclooxygenase (cyclooxygenase or prostaglandin H2 synthetase; hereinafter called COX) -2 enzymes by external stimuli such as ultraviolet rays (Wilgus et al., 2000. Prostaglandins). & other Lipid Mediators, Vol. 62, pp 367-384). Therefore, 1 × 10 ⁴ keratinocytes were placed in each well of a 96-cell culture plate for COX-2 enzymatic activity inhibition experiment in which arachidonic acid in the cell membrane was converted to prostaglandin due to UV damage. Attached for hours. Aspirin was treated to the final keratinocytes to a final concentration of 500 uM to inhibit the activity of prostaglandin biosynthetic enzymes present in the keratinocytes. After 2 hours of aspirin treatment, each well containing keratinocytes was washed twice with phosphate buffered saline (PBS) and 50 μl of phosphate buffered saline (PBS) was added to each well. The keratinocytes were irradiated with UV 30mJ / cm 2 using an ultraviolet B lamp (Model: F15T8, UV B 15W, Sankyo Dennki, Japan), and then PBS was removed and each cell was prepared with keratinocyte culture medium 200. Μl was added. The fructose 1,6-diphosphate was treated here at concentrations of 20 mM, 10 mM and 5 mM, and then incubated for 16-20 hours.

The prostaglandin E2 (PGE2) biosynthesized and accumulated in the culture supernatant was quantified to determine the effect of PGE2 inhibition of fructose 1,6-diphosphate. The amount of PGE2 was quantified using an enzyme immunoassay, and the results are shown in FIG. 4.

As shown in Figure 4, it can be seen that fructose 1,6-diphosphate reduced the amount of PGE2 biosynthesized from COX-2 enzyme by ultraviolet light. Ultraviolet (UV B) stimulation generally induces an inflammatory response that increases the blood flow and vascular permeability of the skin, resulting in edema and erythema, and slowly causing cells such as neutrophils to PGE2 increases at the same time as the stimulus, increasing inflammatory factors that attract immune cells to the site of inflammation (TAWilgus et al., Topical application of a selective cyclooxygenase inhibitor suppresses UVB mediated cutaneous inflammation, Prostaglandins & other lipid mediators 62 , 367-384, 2000). This demonstrates that fructose 1,6-diphosphate has an anti-inflammatory effect that reduces the increase in inflammatory mediators caused by UV light.

[Test Example 5] Skin irritation test at the animal level

In order to examine the degree of skin irritation of fructose 1,6-diphosphate, rabbits were tested for the skin irritation by the following method. Rabbits (2 to 2.5 kg male New Zealand white rabbits), freely fed water and solid food, and adapted to the environment were used for the test. After shaving the back hair, the sample and the control material were applied to a size of about 1.5 cm x 1.5 cm. The area that can be applied per rabbit is 8 to 10 sites. The test substance is applied to the animal administration site twice a day for 6 hours, and the applied site is exposed to air. The applied site was observed twice a day immediately before the next application, and the presence or absence of irritation such as erythema, skin formation, edema formation, and the degree thereof was evaluated according to the criteria shown in Table 1 below. The results are shown in Table 2 below, and the stimulation indexes shown in Table 2 are the results of the method of calculating the primary stimulation index of the commonly used Draize (Draize, JH, Appraisal of the safety of chemical in foods, drugs and cosmetics.The staff of the division of pharmacology, Food and Drug Education and Welfare, Pub. 1959), proving that fructose 1,6-diphosphate has no irritation compared to other cosmetics As a comparative substance, vitamin C, an antioxidant used in cosmetics, was used.

Degree of skin irritation grade 1) No erythema and crust-forming erythema Very mild erythema (only visible to the naked eye) Clear erythema Strong erythema Deep erythema Strong erythema and crust formation 01234 2) No edema forming edema Very mild edema (only visible to the naked eye) Clear edema (distinguishable from clear) Moderate edema (if swollen up to 1 mm) Strong edema (swelling more than 1 mm and exposed area) Extended out) 01234 * Primary irritation index (P.I.I.) = (Average of sum of erythema rating and edema rating) / 4

Skin Primary Irritation Index (0 ~ 4) Comparative group (20% aqueous solution of vitamin C) 2.5 Fructose 1,6-diphosphate 20% aqueous solution 1.0 Aqueous solution 0.7

As can be seen in Table 2, the fructose 1,6-diphosphate can be seen that there is no skin irritation compared to the conventional antioxidant vitamin C.

Test Example 6 Effect of Fructose 1,6-diphosphate on Skin Erythema

The effects of fructose 1,6-diphosphate on UV-induced erythema-induced erythema-induced effects in healthy volunteers who had not taken nonsteroidal anti-inflammatory drugs and other steroidal agents for more than a month prior to the experiment were examined by Kaidbey and Kurban. Evaluation was made according to the method (Kaidbey & Kurban, 1976, J. Invest. Dermatol., Vol 66 pp153-156). After attaching an opaque tape with six holes 1.5cm in diameter to the upper forearm of the subjects, 12 healthy men were exposed to UV (UV B) twice as much as the minimum ErythemaDose of each subject. The erythema was induced by irradiation with a lamp (wavelength 290-320 nm, Toshiba). Each experimental formulation was prepared according to the formulation of Comparative Examples and Examples of Table 3 below, and was applied 1 hour before and after UV irradiation. The results are shown in Table 4 below, and the erythema index shown in Table 4 was evaluated 6 hours after and 24 hours after UV irradiation according to the criteria shown in Table 1 above.

ingredient Content (% by weight) Control Example Comparative example Fructose 1,6-diphosphate - 1.00 - Vitamin C - - 5.00 Beeswax 4.00 4.00 4.00 Polysorbate 1.50 1.50 1.50 Sorbitan sesquioleate 0.70 0.70 0.70 Liquid paraffin 5.00 5.00 5.00 Caprylic / Capric Triglycerides 5.00 5.00 5.00 glycerin 3.00 3.00 3.00 Butylene glycol 3.00 3.00 3.00 Propylene glycol 3.00 3.00 3.00 Carboxy Vinyl Polymer 0.10 0.10 0.10 Triethanolamine 0.20 0.20 0.20 antiseptic Quantity Quantity Quantity Pigment Quantity Quantity Quantity Spices Quantity Quantity Quantity Purified water to 100 to 100 to 100

Inhibitory Effect of Fructose 1,6-diphosphate on Skin Erythema by UV Light Experimental group Redness Index (2MED) 6 hours 24 hours Control 1.7 2.2 Example 0.9 1.6 Comparative example 1.6 2.4

As can be seen in Table 4, the cosmetic containing fructose 1,6-diphosphate was found to inhibit the erythema induced by ultraviolet light. In other words, the cosmetics containing fructose 1,6-diphosphate was found to contribute to maintaining the antioxidant properties of the skin function by inhibiting skin cell damage caused by ultraviolet light.

As described above, the cosmetic composition of the present invention contains fructose 1,6-diphosphate and salts thereof and does not irritate the skin, and is excellent in suppressing skin cell damage caused by external stimuli such as ultraviolet rays. . Therefore, fructose 1,6-diphosphate can be used as a cosmetic raw material that serves as an endogenous antioxidant that increases the self-reducing power of skin cells, as found in the present invention.

Claims (4)

Fructose 1,6-diphosphate (D-fructose 1,6-diphosphate) and the salt thereof is a cosmetic composition for inhibiting skin cell damage, characterized in that it contains an amount of 0.0001 to 20% by weight based on the total weight of the composition. The cosmetic composition according to claim 1, wherein the skin cell damage is skin cell damage caused by ultraviolet rays. The cosmetic composition according to claim 1, wherein the skin cell damage is skin aging. A cosmetic composition for inhibiting skin cell damage by containing fructose 1,6-diphosphate and salts thereof, thereby inhibiting the activity of cyclooxygenase-2 to reduce the concentration of inflammatory mediators of prostaglandins.