COMPOSITION FOR PREVENTING OR IMPROVING UV-INDUCED SKIN DAMAGE USING HYDROANGENOL AS ACTIVE INGREDIENT

Abstract

Provided is a composition for preventing or improving UV-induced skin damage that contains hydrangenol derived from the extract of Hydrangea serrata as an active ingredient. The composition containing hydrangenol derived from the extract of Hydrangea serrata is able to increase secretion of hyaluronic acid and procollagen type-1 and inhibit secretion of MMP-1 in skin cells, and therefore effective in preventing or improving UV-induced damage of skin cells. Accordingly, the hydrangenol-containing composition is usefully available as a quasi-drug, drug, food, or cosmetic composition.

Claims

1. A cosmetic composition for preventing or improving UV-induced skin damage, the composition comprising hydrangenol represented by the following chemical formula 1 as an active ingredient: ##STR00003##

2. (canceled)

3. The cosmetic composition as claimed in claim 1, wherein the hydrangenol is isolated from an extract of leaf of Hydrangea serrata.

4. The cosmetic composition as claimed in claim 3, wherein the hydrangenol is obtained by ethanol extraction of leaf of Hydrangea serrata, ethanol or methanol solvent fractionation, and recrystallization.

5. The cosmetic composition as claimed in claim 1, wherein the hydrangenol is contained in an amount of 0.0001 to 10 wt. % with respect to the total weight of the composition.

6. The cosmetic composition as claimed in claim 1, wherein the hydrangenol accelerates proliferation of skin cells damaged by UV radiation, reduces production of MMP-1, and increases production of procollagen and hyaluronic acid.

7. The cosmetic composition as claimed in claim 1, wherein the composition is used to moisturize the skin irritated by UV radiation or improve wrinkles.

8. (canceled)

9. The cosmetic composition as claimed in claim 1, wherein the composition is for topical administration on the skin and has at least one dosage form selected from the group consisting of toner, essence, nutrition cream, moisturizing cream, gel, lotion, and ointment.

10. (canceled)

11. The cosmetic composition as claimed in claim 3, wherein the composition is for topical administration on the skin and has at least one dosage form selected from the group consisting of toner, essence, nutrition cream, moisturizing cream, gel, lotion, and ointment.

12. The cosmetic composition as claimed in claim 4, wherein the composition is for topical administration on the skin and has at least one dosage form selected from the group consisting of toner, essence, nutrition cream, moisturizing cream, gel, lotion, and ointment.

13. The cosmetic composition as claimed in claim 5, wherein the composition is for topical administration on the skin and has at least one dosage form selected from the group consisting of toner, essence, nutrition cream, moisturizing cream, gel, lotion, and ointment.

14. The cosmetic composition as claimed in claim 6, wherein the composition is for topical administration on the skin and has at least one dosage form selected from the group consisting of toner, essence, nutrition cream, moisturizing cream, gel, lotion, and ointment.

15. The cosmetic composition as claimed in claim 7, wherein the composition is for topical administration on the skin and has at least one dosage form selected from the group consisting of toner, essence, nutrition cream, moisturizing cream, gel, lotion, and ointment.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0028] FIG. 1 is a schematic diagram showing the separation and purification process of preparing hydrangenol from the leaves of Hydrangea serrata.

[0029] FIG. 2 is an ESIMS (positive-ion mode) spectrum of hydrangenol.

[0030] FIG. 3 is a .sup.1H-NMR spectrum of hydrangenol.

[0031] FIG. 4 is a .sup.13C-NMR spectrum of hydrangenol.

[0032] FIG. 5 is a DEPT NMR spectrum of hydrangenol.

[0033] FIG. 6 is a HSQC NMR spectrum of hydrangenol.

[0034] FIG. 7 is a HMBC NMR spectrum of hydrangenol.

[0035] FIGS. 8A to 8D are a graph comparing the cell proliferation rate of the solvent-specific extract of Hydrangea serrate in epidermal and dermal cells with UV-induced damage (392-45A: Hydrangea serrata, EGCG: positive control, HaCaT: epidermal cell, and Hs68: dermal cell).

[0036] FIG. 9 is a graph showing the cell proliferation rate of hydrangenol in epidermal and dermal cells with UV-induced damage.

[0037] FIGS. 10A to 10D are a graph comparing the inhibitory effect of the solvent-specific extracts of Hydrangea serrata against MMP-1 in epidermal and dermal cells damaged by UV exposure.

[0038] FIG. 11 is a graph showing the inhibitory effect of hydrangenol against MMP-1 in epidermal and dermal cells damaged by UV exposure.

[0039] FIGS. 12A and 12B area graph comparing the production yield of procollagen by the action of the solvent-specific extracts of Hydrangea serrata in dermal cells damaged by UV exposure.

[0040] FIG. 13 is a graph showing the production yield of procollagen by the action of hydrangenol in dermal cells damaged by UV exposure.

[0041] FIGS. 14A to 14D are a graph showing the production yield of hyaluronic acid by the action of the solvent-specific extracts of Hydrangea serrata in epidermal and dermal cells damaged by UV exposure.

[0042] FIG. 15 is a graph showing the production yield of hyaluronic acid by the action of hydrangenol in epidermal and dermal cells damaged by UV exposure.

BEST MODES FOR CARRYING OUT THE INVENTION

[0043] Hereinafter, the present invention will be described in further detail with reference to examples. It will be obvious to those skilled in the art that these examples are illustrative purposes only and are not construed to limit the scope of the present invention.

EXAMPLE 1

Preparation of Extract of Hydrangea Serrata

[0044] The extract of Hydrangea serrata in the composition of the present invention was prepared in the following steps. Firstly, leaves of Hydrangea serrate were botanized in Jeju Island (South Korea), dried out for 4-5 days, and chopped to obtain a material for extraction. 25 g of chopped Hydrangea serrata was subjected to reflux extraction in hot water and 175 ml (7-fold, v/v) of ethanol (30%, 50%, 70%) at 50 C. for 3 hours. The product obtained by extraction was removed of insoluble substances through a Whatman (No 2.) extractant filter paper. Then, the product was concentrated under reduced pressure in a distillation apparatus equipped with a condenser and completely removed of the solvent. The extract of Hydrangea serrata thus obtained was dried out to an extraction yield of 20%.

EXAMPLE 2

Preparation of Hydrangenol Derived from Extract of Hydrangea Serrata

[0045] 5.66 g of the 70% ethanol extract obtained in Example 1 was subjected to a gel filtration with a Diaion HP-20. Each 2 L of the mixed solution of methanol (30%, 50%, 70%, 100%) and CH.sub.2Cl.sub.2MeOH (1:1, v/v) was used as a developing solvent for solvent fractionation into five subfractions (392-70EDia 15). The subfraction 392-70EDia4 (357.4 mg) was solvent-fractionized with Sephadex LH-20 and a developing solvent of methanol into seven subfractions (392-70EDia4a4g). The 392-70EDia4d subfraction was recrystallized in methanol to yield 31.1 mg of an amorphous compound 1 (hydrangenol). An ESIMS (positive-ion mode) analysis conducted to identify the structure of the product in Example 2 revealed that m/z=257[M+H].sup.+ (Refer to FIG. 2). As can be seen from the .sup.1H-NMR spectrum (Refer to FIG. 3), in strong magnetic field, the methane proton (H-3) at H 5.50 formed a vicinal coupling with the methylene proton (H-4) at H 3.30 and 3.06. The chemical shift value as well as the vicinal coupling rendered the protons originated from the C-ring. As for the protons originated from the p-substituted benzene ring of a B-ring, the peaks H-2 and H-3 and the peaks H-6 and H-5 formed an ortho-coupling and showed up as a doublet (J =8.4 Hz); and the peaks H-2 and H-6 and the peaks H-3 and H-5 also formed an ortho-coupling and showed up as a doublet. This indicated the chemical structure rendered symmetric with respect to the hydroxyl group. In the 1,2,3-trisubstituted benzene of the A-ring, the protons H-5 and H-7 independently formed a coupling with the proton H-6, and an ortho-coupling was formed between the protons H-5 and H-7, which appeared as a doublet. The proton H-6 made an ortho-coupling and a meta-coupling and showed up as a double of doublets. This revealed that all the peaks corresponded to one proton.

[0046] In the .sup.13C-NMR spectrum (Refer to FIG. 4), fifteen peaks including a para-substituent appeared. The quaternary carbon peak at C 172 was originated from the first carbon of the compound, that is, the carbonyl group; the peaks at C 116.9 (C-3, C-5) and C 129.6 (C-2, C-6) were originated from the para-substituent of an aromatic ring; and the peaks at C 36.1 and C 83.1 were originated from an aliphatic carbon and an oxygenated carbon, respectively. In the DEPT NMR spectrum (Refer to FIG. 5), seven protonated carbons were identified and the peak at C 36.1 was a methylene group originated from the C-4.

[0047] A 2D NMR analysis was carried out to analyze the precise structures of the peaks. The precise positions of the peaks were addressed according to the HSQC (Refer to FIG. 6), and the bonding positions of substituents were determined from the HMBC (Refer to FIG. 7). That is, the peak at H 7.26 (2H, d, J=8.4 Hz, H-2, 6) had a correlation with C-4 at C 36.1; whereas the peaks at H 3.06 and H 3.30 originated from H-4 had a correlation with the peaks at C 83.1 (C-3), C 119.8 (C-5), C 110.0 (C-9), and C 142.2 (C-10). A summary of the results and a comparison with the literatures identified the compound of Example 2 as hydrangenol (Yoshikawa M., Matsuda H., Shimoda H., Shimada H., Harada E., Naitoh Y., Miki A., Yamahara J., Murakami N. Development of Bioactive Functions in Hydrangeae Dulcis Folium. V. On the Antiallergic and Antimicrobial Principles of Hydrangeae Dulcis Folium. (2). Thunberginols C, D, and E, Thunberginol G 3-O-Glucoside, ()-Hydrangenol 4-O-Glucoside, and (+)-Hydrangenol 4-O-Glucoside. Chem. Pharm. Bull. 1996, 44: 1440-1447).

EXPERIMENTAL EXAMPLE 1

Effect of Hydrangenol on Proliferation of Cells with UV-Induced Damage

[0048] Samples obtained in Examples 1 and 2 were measured in regards to the effect in recovering the skin damaged from UV-B exposure. In this experiment, HaCaT keratinocytes and HS68 fibroblasts were used as epidermal and dermal cells, respectively. In order to evaluate the possible efficacy of each sample in preventing or improving the UV-induced damage of skin cells, the cells were seeded into a 96-well microplate at a density of 1.010.sup.4 cells/well and stabilized for 24 hours. Next, the culture medium was exchanged to a new one supplemented with the sample, and the cells were incubated for 24 hours. For UV-B irradiation, the culture medium was removed, washed with PBS, and exposed to UV-B radiation at 15 mJ/cm.sup.2. After an incubation of 24 hours in a culture medium supplemented with the sample, the cells were subjected to an MTT assay to measure cell viability. The MTT assay measures the reduction of a tetrazolium component (MTT) into a formazan product by the mitochondria of viable cells. More specifically, 50 l of a 5 mg/ml MTT solution was added to the cells, and after an incubation of 4 hours, the culture solution was completely removed and cells were dissolved in DMSO. The absorbance of the DMSO solution in the microplate was quantified by spectrophotometry at 540 nm.

[0049] As shown in FIG. 8, the extracts of Example 1 was all non-cytotoxic to the epidermal and dermal cells damaged by UV-B radiation of 15 mJ/cm.sup.2 and mostly effective in promoting proliferation of the most of the cells. Especially, the dermal cells treated with the extracts of Example 1 had a similar level of proliferation to those with 10 M epigallocatechin gallate (EGCG) used as a positive control. Taking the EGCG being a single substance under consideration, the extracts of Example 1 presumably contained a beneficial ingredient more effective in improving UV-induced skin damage. As shown in FIG. 9, the hydrangenol of Example 2 was non-cytotoxic and effective in promoting cell proliferation in the epidermal and dermal cells damaged by UV-B radiation of 15 mJ/cm.sup.2. In particular, 1 M hydrangenol was equivalent to 10 M EGCG in the cell proliferation effect. In relation to the EGCG with a concentration of 10 M, the hydrangenol, even with a low concentration, was able to make an excellent effect in preventing or improving UV-induced skin damage.

EXPERIMENTAL EXAMPLE 2

Quantitative Analysis of MMP-1

[0050] Samples obtained in Examples 1 and 2 were measured in regards to the inhibitory effect against the secretion of MMP-1 in epidermal HaCat keratinocytes and dermal Hs68 fibroblasts. In order to evaluate the effect of each sample in reducing MMP-1, the cells were seeded into a 24-well microplate at a density of 1.010.sup.5 cells/well and stabilized for 24 hours. Next, the culture medium was exchanged to a new one supplemented with the sample, and the cells were incubated for 24 hours. For UV-B irradiation, the culture medium was removed, washed with PBS, and exposed to UV-B radiation at 15 mJ/cm.sup.2. After an incubation of 48 hours in a culture medium supplemented with the sample, the resultant supernatant was measured in regards to the degree of secretion of MMP-1 by using an MMP-1 Human ELISA kit (ab100603, abcam, US).

[0051] As shown in FIG. 10, all the extracts of Example 1 reduced the production of MMP-1 in a concentration-dependent manner in the epidermal and dermal cells damaged by UV-B radiation of 15 mJ/cm.sup.2. As shown in FIG. 11, the hydrangenol of Example 2 also inhibited the production of MMP-1 in a concentration-dependent manner in the epidermal and dermal cells damaged by UV-B radiation of 15 mJ/cm.sup.2. Particularly, in the dermal cells, the hydrangenol was far superior to the EGCG at a same concentration in terms of the inhibitory effect against the production of MMP-1. Therefore, it was implied that the hydrangenol of Example 2 was effective in improving UV-induced skin damage by inhibiting the production of MMP-1 incurred by UV exposure.

EXPERIMENTAL EXAMPLE 3

Analysis of Procollagen Type-1 Content

[0052] Samples obtained in Examples 1 or 2 were measured in regards to the effect of increasing procollagen type 1 in dermal Hs68 fibroblasts. In order to evaluate the effect of each sample in increasing secretion of procollagen type 1, the cells were seeded into a 24-well microplate at a density of 1.010.sup.5 cells/well and stabilized for 24 hours. Next, the culture medium was exchanged to a new one supplemented with the sample, and the cells were incubated for 24 hours. For UV-B irradiation, the culture medium was removed, washed with PBS, and exposed to UV-B radiation at 15 mJ/cm.sup.2. After an incubation of 48 hours in a culture medium supplemented with the sample, the resultant supernatant was measured in regards to the degree of secretion of procollagen type 1 by using a Procollagen type 1 C-peptide (PIP) EIA kit (Mk101, Takara, Japan).

[0053] As shown in FIG. 12, all the extracts of Example 1 increased the production of procollagen in the dermal cells damaged by UV-B radiation of 15 mJ/cm.sup.2. 50% and 70% extracts were particularly superior to a single substance, EGCG, in the efficacy of procollagen production (Refer to FIG. 12-b). On account of this, the extracts of Example 1 presumably contained a beneficial ingredient for skin elasticity. As shown in FIG. 13, the hydrangenol of Example 2 increased the production of procollagen in the dermal cells damaged by UV-B radiation of 15 mJ/cm.sup.2. In particular, 1 M hydrangenol was equivalent to 10 M EGCG in the procollagen production effect. In relation to the EGCG with a concentration of 10 M, the hydrangenol, even with a low concentration, was able to make an excellent effect in promoting production of procollagen. Therefore, the hydrangenol of Example 2 proved to increase the production of procollagen and aid the regeneration of collagen fibers degraded by UV radiation, thereby preventing or improving UV-induced skin damage.

EXPERIMENTAL EXAMPLE 4

Analysis of Hyaluronic Acid Content

[0054] Samples obtained in Examples 1 and 2 were measured in regards to the effect of increasing hyaluronic acid in epidermal HaCaT keratinocytes and dermal Hs68 fibroblasts. In order to evaluate the effect of each sample in increasing hyaluronic acid, the cells were seeded into a 24-well microplate at a density of 1.010.sup.5 cells/well and stabilized for 24 hours. Next, the culture medium was exchanged to a new one supplemented with the sample, and the cells were incubated for 24 hours. For UV-B irradiation, the culture medium was removed, washed with PBS, and exposed to UV-B radiation at 15 mJ/cm.sup.2. After an incubation of 24 hours in a culture medium supplemented with the sample, the resultant supernatant was measured in regards to the degree of secretion of hyaluronic acid by using a TECO Hyaluronic Acid PLUS ELISA kit (TE 1018-2, TECO Medical Group, US).

[0055] As shown in FIG. 14, all the extracts of Example 1 increased the production of hyaluronic acid in the epidermal and dermal cells damaged by UV-B radiation of 15 mJ/cm.sup.2. Particularly in the dermal cells, most of the extracts, even with the lowest concentration, showed an excellent effect of promoting hyaluronic acid. As shown in FIG. 15, the hydrangenol of Example 2 also increased the production of hyaluronic acid in the epidermal and dermal cells damaged by UV-B radiation of 15 mJ/cm.sup.2. In particular, 1 M hydrangenol exhibited an excellent effect in producing hyaluronic acid. Accordingly, the hydrangenol of Example 2 proved to increase the production of hyaluronic acid and aid the moisturization of the skin, thereby preventing or improving UV-induced skin damage.

FORMULATION EXAMPLE 1

Preparation of Tablets

[0056] The extract of Example 2 was mixed with the ingredients of Table 1 and processed into tablets according to a general preparation method for tablet.

TABLE-US-00001 TABLE 1 Ingredients Unit weight (mg) Example 2 10 Corn starch 100 Lactose 100 Stearic acid 2

FORMULATION EXAMPLE 2

Preparation of Capsules

[0057] The extract of Example 2 was mixed with the ingredients of Table 2 and filled in gelatin capsules to prepare soft capsules according to a general preparation method for capsule.

TABLE-US-00002 TABLE 2 Ingredients Unit weight (mg) Example 2 2 Vitamin E 2.25 Vitamin C 2.25 Palm oil 0.5 Vegetable hydrogenated oil 2 Yellow lead 1 Lecithin 2.25 Filling solution for soft capsule 387.75

FORMULATION EXAMPLE 3

Preparation of Liquid

[0058] The extract of Example 2 was mixed with the ingredients of Table 3 and filled in a bottle or a pouch to prepare a liquid according to a general preparation method for beverage.

TABLE-US-00003 TABLE 3 Ingredients Unit weight (g) Example 2 0.0205 Xanthan gum 0.0075 Pructooligosaccharide 0.7500 Powdered coconut flower nectar 1.0500 Concentrated ssangwha-tang 1.5000 Red ginseng flavor 0.0450 Purified water 20.1425 Filling solution for soft capsule 387.75

FORMULATION EXAMPLE 4

Preparation of Chewable Gel

[0059] The extract of Example 2 was mixed with the ingredients of Table 4 and filled in a three-sided seal pouch to prepare a chewable gel according to a general preparation method for chewable gel.

TABLE-US-00004 TABLE 4 Ingredients Unit weight (g) Example 2 0.0200 Food gel 0.3600 Carrageenan 0.0600 Calcium lactate 0.1000 Sodium citrate 0.0600 Complex scutellaria extract 0.0200 Enzymatically modified stevia 0.0440 Fructooligosaccharide 5.0000 Red grape concentrate 2.4000 Purified water 13.9560

FORMULATION EXAMPLE 5

Preparation of Chewable Gel

[0060] The extract of Example 2 was processed into the composition of Table 5 according to a general preparation method for nutrient cream.

TABLE-US-00005 TABLE 5 Ingredients Content (%) Example 2 0.01 Sitosterol 4.0 Polyglyceryl 2-oleate 3.0 3.0 Ceteareth-4 2.0 Cholesterol 3.0 Dicetyl phosphate 0.4 Concentrated glycerin 5.0 Sunflower oil 22.0 Carboxylvinyl polymer 0.5 Triethanol amine 0.5 Preservative trace Flavor trace Purified water balance

[0061] The above-defined composition is given as a formulation example using a mixture of appropriate compositions. Yet the mixing ratio and the ingredients maybe varied arbitrarily under necessity.

[0062] The extract of the present invention was stable under the testing conditions for all formulation examples and hence not problematic in the stability of the dosage form.

INDUSTRIAL AVAILABILITY

[0063] As described above, the composition containing hydrangenol derived from the extract of Hydrangea serrata according to the present invention is able to reduce secretion of MMP-1 produced by UV-B exposure and increase secretion of hyaluronic acid and procollagen type-1, thereby preventing UV-induced skin aging and maintaining skin elasticity. Accordingly, the composition of the present invention is usefully available as a drug, food or cosmetic composition.