COSMETIC COMPOSITIONS FOR PROTECTION AGAINST LIGHT-INDUCED DAMAGES

Abstract

The invention relates to cosmetic compositions [compositions (C)] comprising an oak extract, a grape seed extract and a green tea extract and to the use of a composition (C) to protect body parts exposed to light against damages. It further relates to a cosmetic method [method (CM)] for protecting body parts exposed to light against damages, said method comprising applying a composition (C) to a body part exposed to light.

Claims

1. A method of protecting body parts from damage induced by light radiation, the method comprising the step of: application of a cosmetic composition comprising an oak extract, a grape seed extract and a green tea extract to body parts exposed to the light radiation.

2. The method of claim 1, wherein the light radiation has a wavelength of from 315 to 400 nm (UV-A) and/or a wavelength from 400 to 495 nm.

3. The method of claim 2, wherein the light radiation has a wavelength of from 400 to 495 nm.

4. The method of claim 1, wherein the body part is the skin, the scalp, the hair and/or external mucosae.

5. The method of claim 4, wherein the body part is the skin.

6. The method of claim 1, wherein the oak extract has a total polyphenol content ranging from 30% to 60% w/w.

7. The method of claim 1, wherein the oak extract is present in the cosmetic composition at from 0.01% to 5% w/w.

8. The method of claim 1, wherein the grape seed extract has a total proanthocyanidin content, as calculated by the Folin method and expressed as as catechins, equal to or greater than 95% w/w, and has a monomer content, resulting from the sum of catechin and epicatechin expressed as catechin, ranging from 5% to 15% w/w.

9. The method of claim 1, wherein the grape seed extract is present in the cosmetic composition at from 0.01% to 5% w/w.

10. The method of claim 1, wherein the green tea extract has a polyphenol content, as calculated by the Folin method and expressed as catechins, equal to or greater than 40% w/w and has a catechin content, as expressed as epicatechin-3-O-gallate, equal to or greater than 15% w/w.

11. The method of claim 1, wherein the green tea extract is present in the cosmetic composition at from 0.01% to 5% w/w.

12. The method of claim 1, wherein the damage induced by light radiation is selected from: xerotic skin in inflammatory skin disorders, skin ageing and skin color changes.

13. The method of claim 1, wherein the cosmetic composition further comprises one or more cosmetically acceptable excipients.

14. The method of claim 11, wherein the green tea extract is present in the cosmetic composition at from 0.05% to 1% w/w.

15. The method of claim 1, wherein the green tea extract is present in the cosmetic composition at 0.25% w/w.

16. The method of claim 1, wherein the green tea extract is present in the cosmetic composition at 0.1% w/w.

Description

EXPERIMENTAL SECTION

[0034] Evaluation of the Protective Efficacy of a Composition (C-1) against Blue Light (460 nm)—Induced Protein Oxidative Damage (Carbonylation)

[0035] Outline of the Experimental Procedure

[0036] Human keratinocytes (HaCAT) were irradiated in the presence or absence of a composition (C-1) comprising equal weight amounts of extracts (Q), (GS) and (GT) (Vitakelox™) using an irradiation system available from OxiProteomics®. A solution of N-acetyl-cysteine was used as positive control of protection. Just after irradiation, proteins were extracted from the cells and analyzed. Carbonylated proteins were labeled with specific functionalized fluorescent probes and the resulting samples were resolved by high-resolution electrophoresis. Total proteins were post-stained with Sypro™ Ruby protein gel stain. A Carbonyl (protein damage) Score was obtained for each sample after normalization of the fluorescent signal for carbonylated proteins by total proteins. A significant increase in protein oxidative damage was observed upon blue light irradiation. This increase was prevented in the presence of composition (C-1) at two different concentrations (0.01% and 0.005% w/v) and with two times of treatment (6 hours and 24 hours).

[0037] Detailed Experimental Procedure

[0038] a) Cell Culture and Experimental Design

[0039] HaCaT cells were cultured in calcium-free DMEM (Dulbecco's Modified Eagle Medium), with 10% SVF (stromal vascular fraction), at 37° C. and humid atmosphere, supplemented with 5% CO.sub.2. Cells were seeded (500.000 cells/well) at day 0 (D0) in multi-well plates containing culture medium and distributed in 8 experimental groups as depicted in Table 1.

TABLE-US-00001 TABLE 1 Sampling Group Reference Replicates (time) G-1 Control (not irradiated) 3 Day 2 G-2 Stressed* 3 Day 2 G-3 Composition (C-1), 0.01%, 6 hours 3 Day 2 G-4 Composition (C-1), 0.01%, 24 hours 3 Day 2 G-5 Composition (C-1), 0.05%, 6 hours 3 Day 2 G-6 Composition (C-1), 0.05%, 24 hours 3 Day 2 G-7 Internal reference**, 6 hours 3 Day 2 G-8 Internal reference**, 24 hours 3 Day 2 *stress was induced by irradiation (see point c) below. **N-acetyl-cysteine

[0040] b) Application of Composition (C-1)

[0041] On day 1 (D1), two solutions of composition (C-1) having two different concentrations (0.01% and 0.005%) were prepared by solubilisation of the composition in the culture medium. Experimental groups 4 and 6 were incubated for 24 hours (t2) with the solutions prior to irradiation. At day 2 (D2), experimental groups 3 and 5 were incubated for 6 hours (t1) prior to blue light irradiation. Groups 7 and 8 were incubated with a solution of NAC (N-acetyl-cysteine) for 6 and 24 hours.

[0042] c) Blue Light Irradiation and Sampling

[0043] On day 2 (D2), the cells were washed and a stress was induced by irradiation with Blue Light (LED source, emission peak at λ=460 nm), using an OxiProteomics® irradiation system. Group 1 was not irradiated. Just after irradiation, cells were collected, snap-frozen and stored at −80° C. for the analysis.

[0044] Analysis

[0045] a) Carbonyl View (In-Situ Visualization)

[0046] After treatments with composition (C-1) and irradiation, the cells were fixed by using a mix of 95% ethanol and 5% acetic acid. Carbonylated proteins were labelled by using a specific functionalized fluorescent probe. The total proteins were labelled using Cy3 NHS (N-hydroxysulfosuccinimide) fluorescent probe, which is routinely used in proteomics studies (see, for instance J Chromatogr B Analyt Technol Biomed Life Sci. 2011 May 15; 879(17-18):1439-43). Images were collected using an epifluorescence microscope and analysed with the software imageJ (Rasband, W. S., ImageJ, U.S. National Institutes of Health, Bethesda, Md., USA, http://imagej.nih.gov/ij/, 1997-2014). The comparison between the different conditions was achieved with strictly identical exposure time, focus and resolution.

[0047] b) Carbonyl Score Analysis

[0048] Proteins were extracted from cells, quantified by the Bradford method and split into equal amounts for analyses. The carbonylated proteins were labelled with specific functionalized fluorescent probes and the samples were resolved by high-resolution electrophoresis separation. Total proteins were post-stained with SyproRuby™ protein gel stain. Image acquisition for carbonylated and total proteins was performed using the Ettan® DIGE imager (GE Healthcare). Image processing and analysis was performed using ImageJ (Rasband, W. S., ImageJ, U.S. National Institutes of Health, Bethesda, Md., USA, http://imagej.nih.gov/ij/, 1997-B014). Statistical analyses were accomplished using GraphPad Software (La Jolla, Calif., USA).

[0049] Results

[0050] Carbonyl View

[0051] The in-situ oxidation level (carbonylation) was represented as the superposition of oxidative specific signal (red) and total protein signal (green). Differences in the specific oxidative protein patters were observed for the condition “control” and “irradiated”. The concentration of fluorescent probes was in excess ensuring an exhaustive labelling of proteins. Image collection was conducted in strictly identical conditions (focus, zoom, resolution, exposure time). The presence of composition (C-1) decreased blue light induced oxidation.

[0052] Carbonyl Score

[0053] After extraction and solubilisation, proteins in samples were quantified by the Bradford method using calibrated BSA (bovine serum albumin) as standard (Bradford M. Anal. Biochem., 72, 248, 1976).

[0054] After labelling carbonylated proteins with fluorescent probes, the proteins were resolved onto 4-20% gradient SDS-PAGE. Proteins were fixed to the gel and the carbonylated proteins were evidenced by fluorescence scanning. Total proteins were post-stained with SyproRuby.

[0055] Densitometric analysis of protein bands was performed using ImageJ analysis software (NIH, USA).

[0056] Quantification was obtained from each sample, both for carbonylated and total proteins. Carbonylated protein signal was normalized by total protein signal for each sample in order to obtain the Carbonyl Score.

[00001]$\mathrm{Carbonyl}\mathrm{Score}\left(\mathrm{sample}X\right)=\frac{\mathrm{carbonylated}\mathrm{prot}.\mathrm{fluorescent}\mathrm{signal}\left(\mathrm{sample}X\right)}{\mathrm{total}\mathrm{prot}.\mathrm{fluorescent}\mathrm{signal}\left(\mathrm{sample}X\right)}$

[0057] Carbonyl Score values were quantified for each sample and the average values were also calculated for each experimental condition taking into consideration the replicates; the results are reported in Table 2 here below.

TABLE-US-00002 TABLE 2 Carbonyl Score Group (G) and replicate (R) Oxidized Total Carbonyl number proteins Proteins Score Condition G-1(R1) 11833.33 21509.91 0.550 Control G-1(R2) 11082.24 18175.04 0.610 G-1(R3) 11995.13 19558.61 0.613 G-2 (R1) 13293.93 16795.54 0.792 Stressed G-2 (R2) 14562.57 18606.68 0.783 G-2 (R3) 18147.06 20767.23 0.874 G-3 (R1) 15145.65 17046.06 0.889 Composition (C-1), G-3 (R2) 12443.48 16265.65 0.765 0.01% 6 h G-3 (R3) 12097.48 16026.06 0.755 G-4 (R1) 13306.06 17444.77 0.763 Composition (C-1), G-4 (R2) 11831.18 16069.77 0.736 0.01% 24 h G-4 (R3) 11839.48 15942.77 0.743 G-5 (R1) 10470.77 15506.89 0.675 Composition (C-1), G-5 (R2) 9276.77 15245.48 0.608 0.005% 6 h G-5 (R3) 10370.36 16905.18 0.613 G-6 (R1)* 14599.31 16357.18 0.893 Composition (C-1), G-6 (R2) 10036.23 16145.41 0.622 0.005% 24 h G-6 (R3) 10445.06 15188.06 0.688 G-7 (R1) 12101.36 16480.48 0.734 NAC 6 h G-7 (R2) 11379.65 15343.94 0.742 G-7 (R3) 10415.94 15861.06 0.657 G-8 (R1) 9126.82 14273.53 0.639 NAC 24 h G-8 (R2) 11501.94 16425.23 0.700 G-8 (R3) 10859.53 17197.65 0.631

[0058] A significant increase in oxidized proteins was observed upon irradiation with blue light [group (G-2)].

[0059] Composition (C-1) showed a protective effect against blue light-induced oxidative damage on proteins at both concentrations (0.01% and 0.005% w/v) after 24 h of incubation. A statistically significant protection (p<0.01) against blue light-induced damage was observed for composition (C-1) at 0.005% (w/v) at both time points of incubation (6 h and 24 h).

BIBLIOGRAPHY

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