State-of-the-art Light Protection against Reactive Oxygen Species

3-2014 English Edition International Journal for Applied Science Personal Care Detergents Specialties T. Rudolph, S. Eisenberg, J. Grumelard, B. Herzog State-of-the-art Light Protection against Reactive Oxygen Species

T. Rudolph, S. Eisenberg*, J. Grumelard, B. Herzog** State-of-the-art Light Protection against Reactive Oxygen Species Introduction Light, in particular ultraviolet irradiation (UV), but also visible and near infrared irradiation (IR) are the major sources to Abstract This paper aims at a scientific comparison of two substantially different anti-ros test methodologies: the beta-carotene test (BCT test) and the radical status factor test method (RSF method). The photometer-based BCT test uses the light-sensitive and lipophilic probe of betacarotene to be inserted into the formulation. This probe degrades under sun-like irradiation through its ability to quench lipophilic reactive oxygen species (ROS) like singlet oxygen. The RSF method indirectly measures the UV light-triggered formation of hydroxyl radicals that are trapped by a watersoluble spintrap (electron spin resonance technique). Two state-of-the-art light protection actives, Bis-ethylhexyl hydroxydimethoxy benzylmalonate (HDBM), a liquid, stable, non-yellowing, and non-absorbing skin care antioxidant, plus Bis-ethylhexyloxyphenol methoxyphenyl triazine (BEMT), a broadband and photostable UV filter were combined at various ratios in a light protection cream to be tested in both assays. Under solar simulated light in the BCT test, HDBM performed optimal due to its lipophilic nature and its ability to quench lipophilic ROS in the direct neighbourhood of beta-carotene. By contrast BEMT predominates in the RSF test. Here the product performance mainly originates from BEMT s broadband UV absorbance power that covers both, UVB and UVA. In the discussion about relevant antioxidant testing it appears essential to combine diverse protocols into the evaluation strategy. Here two complementary methods are proposed as well as a combination of modern actives that can pass those assays with distinction. generate reactive oxygen species (ROS) such as singlet oxygen, radicals or peroxides. Today s»sun protection systems«are more and more developing into future»light protection systems«. Modern products may provide photostable broadband UVB/A protection against sunburn and photoaging but may also need to counteract all sources for ROS generation while maintaining an optimal skin feel as well as appealing galenic properties such as being white, stable and non-yellowing. Anti-ROS efficacy test methods are countless. Some include irradiation steps while others do not. Some might only measure a specific group of ROS, like radicals, but do not monitor non-radical ROS like singlet oxygen or peroxides. Some involve hydrophilic measurement techniques, others concentrate on the lipophilic environment. (Photo)oxidative stress is a complex process. To a large extent it is linked to the presence of air oxygen, its transformation into reactive species and their chemical reaction with oxidation-sensitive compounds. Light in the presence of a sensitizer often initiates oxidative stress or autoxidation. Air oxygen is remarkably lipophilic and concentrated Fig. 1 Spectrum of the Atlas CPS+ irradiance 10 SOFW-Journal 140 3-2014

on the skin surface (5), so that lipophilic environments such as sebum are in particular prone to autoxidation (6). Regarding this lipophilic aspect we herein describe the BCT assay (beta carotene test) to capture lipophilic ROS like singlet oxygen, lipoperoxides and lipophilic radicals. In comparison the RSF test (radical status factor) is more directed towards hydrophilic ROS with a focus on the hydroxyl radical. Five cosmetic emulsions were tested for their overall antioxidant performances in both assays. Varying levels of»primary«and»secondary«actives were chosen and their impact on the overall antioxidant protection level investigated. Bis-ethylhexyloxyphenol methoxyphenyl triazine (BEMT (1)), a broadband and photostable UV filter served as»primary«performance active. It was combined with its»secondary«active counterpart Bis-ethylhexyl hydroxydimethoxy benzylmalonate (HD- BM (2, 3)). HDBM is a liquid, stable, non-yellowing, and non-absorbing skin care antioxidant. Materials and methods a) BCT test (beta-carotene test) A solution of 0.5 % (w/w) beta-carotene (Fluka, Art. Nr. 22040) in o-xylene was freshly prepared and 2µLcm -2 of the solution homogeneously applied over the roughened side of a PMMA plate (PMMA plates from Schönberg, sandblasted, 2µm roughness). The plate was allowed to dry (RT, 10 min) before the formulation was applied onto the plate with a positive displacement pipette (2µLcm -2 ) and homogeneously distributed with a finger. In total 8 replicates per product were prepared. 4 replicates were stored in the dark and 4 replicates were subjected to the irradiation step. As irradiation source we used an Atlas CPS+ solar simulator equipped with a water cooled sample tray. As an irradiation dose we applied 150kJm -2 (total UVB plus UVA) corresponding to an erythemal effective dose of about 3 MED. The total irradiance was set to 500Wm -2 of which 58.9 Wm -2 was UV, the remaining visible and IR. From Fig. 1, which shows the irradiance spectrum of the Atlas CPS+, it becomes clear, that the samples in this test Fig. 2 Absorbance in terms of specific extinction of BEMT and HDBM in the spectral range between 290 and 450 nm were exposed to significant amounts of visible radiation. After irradiation the PMMA plates were extracted with isopropanol and diluted to a volume of exactly 50mL. Beta-carotene was photometrically quantified at 452nm. The final recovery of beta-carotene was calculated in comparison to the equally treated dark references. b) Radical Status Factor (RSF) Test This method is based on EPR spectroscopy using a spin probe for the detection of radicals. A spin probe is a stable radical, for instance a nitroxide, which reacts with the radicals induced during UV irradiation, such that the EPR signal of the probe decreases with UV dose (4, 6). Skin biopsies of porcine origin (taken from pig ears) are incubated with a 50/50 water/ethanol 1 mm solution of 3-carboxy-2,2,5,5-tetramethylpyrrolidone-1-oxyl (PCA) for five minutes. Radicals are induced in the skin samples by irradiation with simulated solar UV light (Newport, 300 W; irradiance in the UVB range = 23.5 W/m 2 ). EPR signals are measured after UV doses of 7, 14, 28, 42, and 70 kj/m 2 ), and a rate constant is determined from the decrease of the signal. It is important to note, that the light source used here was comparable in the UV range to the output of the lamp used for the BCT test, but radiation above 450 nm was filtered off with the RSF tests. In order to reduce experimental scatter due to the variability of the biological substrates, a calibration curve is measured for each set of skin samples originating from the same animal, using neutral density filters with known transmission, and thus known protection factor (RSF). The rate constant of the degradation process of the spin probe is determined from the experimental data obtained after the different UV doses, and is normalized to its value without protection (7). The calibration curve is constructed by plotting the normalized rate constants against the RSF values of the neutral density filters. Test formulations are assessed by distributing them at a rate of 2mg/cm 2 on pig skin substrates. After 15 minutes normalized rate constants are determined from the decrease of the EPR signal as function of UV dose. The value of the RSF can then be read from the calibration curve. Each formulation was measured on four skin biopsies. The RSF tests were carried out at Gematria Test Lab GmbH, Berlin, Germany, using an X-band EPR spectrometer Miniscope MS300 (Magenttech GmbH, Berlin, Germany). c) Test formulations Oil-in-water cream containing various amounts of Bis-ethylhexyl hydroxydimethoxy benzylmalonate (HDBM, RonaCare AP, Merck KGaA) and Bis-ethylhexyloxyphenol methoxyphenyl triazine (BE- SOFW-Journal 140 3-2014 11

MT, Tinosorb S, BASF SE) were prepared. The compositions are listed in Table 1. Fig. 2 shows results of UV spectroscopic measurements of BEMT and HDBM in terms of specific extinction (E11, absorbance at a concentration of 1 % (w/v) and an optical path length of 1 cm) in the spectral range between 290 and 450 nm. While BEMT is efficient in absorbing UV over a broad spectral range, there is no UV attenuation by HDBM. Results and discussion In this study two types of tests were used, the BCT test (beta carotene test) and the RSF test (radical status factor test). From the differences in testing conditions, complementary results can be expected. The main differences in the test conditions are summarized in Table 2. a) BCT test (Beta carotene test) Carotenoids, mainly beta-carotene (Provitamin A) and lycopene are part of the natural skin s defense system against light-induced oxidative stress (8). Carotenoids are ingested through the diet and can reach the outermost skin layers were they are exposed to solar light and can be degraded by light-induced singlet oxygen, lipophilic radicals and/ or lipoperoxides. It is well known that under light exposure beta-carotene can form lipophilic ROS like peroxyl radicals and lipoperoxides that further degrade beta carotene itself. In addition beta-carotene is a highly efficient singlet oxygen quencher. In plants it neutralizes singlet oxygen that is produced in a sensitized reaction by the green leaf pigment chlorophyll. Beta-carotene thus helps to maintain the photosynthesis (9). The quenching of lipophilic ROS leads to beta-carotene s photodegradation as BCT test demonstrated in the BCT test (Fig. 3). Here we used the extreme light-sensitivity of beta-carotene as a suitable criterion to measure the product s ability to prevent beta-carotene from being photooxidized. The photooxidation level may then be transferred to other light-sensitive skin lipids like squalene, unsaturated fatty acids or retinoids (10). The test results indicate (Fig. 3) that in the placebo formulation A) almost 80 % of beta-carotene were photodegraded under sun-simulated light containing a total UV dose of 150kJm -2 (equivalent to 3 MED). The addition of 1 % of the antioxidant HDBM in formulations B), D) and F) drastically reduced the beta-carotene degradation from 80 % back to 15 % in RSF test Substrate PMMA Pig ear skin Irradiation UV + visible UV Nature of probe hydrophobic hydrophilic Table 2 Comparison of BCT- and RSF testing conditions. Trade Name INCI Name A B C D E F Part A Part B Emulgin Prisma (BASF) Disodium Cetearyl Sulfosuccinate 0.50 0.50 0.50 0.50 0.50 0.50 Lanette O (BASF) Cetearyl Alcohol 1.00 1.00 1.00 1.00 1.00 1.00 Cetiol B (BASF) Dibutyl Adipate 5.00 4.00 5.00 4.00 5.00 4.00 X-Tend 226 (ISP) Phenethyl Benzoate 12.00 12.00 12.00 12.00 12.00 12.00 RonaCare AP (MERCK) Bis-Ethylhexyl Hydroxydimethoxy Benzylmalonate 1.00 1.00 1.00 Tinosorb S (BASF) Bis-Ethylhexyloxyphenol Methoxyphenyl Triazine 1.00 1.00 3.00 3.00 Water Aqua 83.88 83.88 82.78 82.78 80.58 80.58 Rhodicare S (Rhodia) Xanthan Gum 0.50 0.50 0.50 0.50 0.50 0.50 Edeta BD (BASF) Disodium EDTA 0.20 0.20 0.20 0.20 0.20 0.20 Part C DC 246 Fluid (Dow Corning) Cyclohexasiloxane (and) Cyclopentasiloxane 4.00 4.00 4.00 4.00 4.00 4.00 Part D Tinovis ADE (BASF) Sodium Acylates Copolymer (and) Hydrogenated Polydecene (and) PPG-1 Trideceth-6 0.40 0.40 0.40 0.40 0.40 0.40 Part E Germall Plus (ISP) Diazolidinyl Urea (and) Iodopropynyl Butylcarbamate 0.15 0.15 0.15 0.15 0.15 0.15 Table 1 Compositions of formulations 12 SOFW-Journal 140 3-2014

The high RSF of 6.7 of emulsion C containing 1 % of BEMT can be explained by the broadband absorption of UV-radiation of this molecule, protecting the skin sample from radical formation. Effects of visible light are not to be expected here, as the Newport solar simulator shows a steep decrease of irradiance at wavelengths above 400nm. Again, the presence of 1 % HDBM in emulsion D) leads only to a small increase of the RSF to 7.7. Conclusion Fig. 3 BCT test (beta-carotene test); six emulsions A) to F) tested for their antioxidative potential; emulsion A) is the control without HDBM or BEMT (UV dose 150kJm -2 ); error bars represent ± standard deviation of four replicates. formulation F). In this assay the influence of the UV absorber BEMT on the photostabilization of beta-carotene was less obvious: In formulation E), 3 % BEMT reduced the beta-carotene photodegradation only from 80 % to about 50 %. One possible mechanistic explanation for the outstanding performance of HDBM in this assay might be the impact of visible light present in the irradiation spectrum (11). The UV absorber BEMT does efficiently absorb broadband UV, but does not absorb visible light. The lipophilic antioxidant HBDM however neutralizes singlet oxygen and other lipophilic ROS irrespectively of the spectral light situation. It hence may extend the broadband UV protection by BEMT into the visible region. reason for the small effect of HDBM could be that it does not penetrate very deep into the skin, where still radicals may have formed. As HDBM does not absorb in the UV/Vis region, the slight increase of the RSF could be explained by small amounts of the spin probe (about 0.5 % of its total concentration) partitioning into the oil phase. Together, both tests, RSF and BCT, cover a broad range of most important parameters in the discussion about relevant anti-ros protection. Firstly they combine lipophilic as well as hydrophilic environments, and thus for instance can take the lipophilic nature of air oxygen into account. Both tests cover radical and non-radical reactive oxygen species of which non-radical singlet oxygen plays an important role at the origin of the radical chain reaction. Finally they include sun simulating UV light conditions comprising not only UV, but also visible and IR as additional source for ROS generation (as lately discovered) (11). b) RSF test results Fig. 4 shows the results of the RSF tests, which have been performed with emulsions A, B, C, and D. The RSF of the placebo formulation (control) without HDBM or BEMT shows as expected a value of 1.0. The RSF of emulsion B) which contains 1 % HDBM is only slightly higher, at 1.4. This can be explained by the fact that the radical probe mainly goes into the water phase of the system (the n-octanol/water partition coefficient of the PCA spin probe is 0.0047 (12)), whereas HDBM is located in the lipophilic phase. Another Fig. 4 RSF results with emulsions A) to D) tested for their antioxidative potential; emulsion A) is the control without BEMT or HDBM (UV dose 70 kjm -2 ); error bars represent ± standard deviation of four replicates. SOFW-Journal 140 3-2014 13

In conclusion it appears essential to combine diverse protocols into the evaluation of the antioxidative potential of cosmetic skin and sun care products. Here two complementary methods, the lipophilic BCT and the hydrophilic RSF test, were proposed as well as the actives combination of HDBM and BEMT that have been shown to pass those assays with distinction. References (1) B. Herzog, D. Hüglin, E. Borsos, A. Stehlin, H. Luther; New UV Absorbers for Cosmetic Sunscreens A Breakthrough for the Photoprotection of Human Skin, Chimia 58, 554 559 (2004) (2) R. Graf, J. Beck, T. Rudolph, K. Jung, T. Herrling, F. Pflücker; Antioxidative Power of Formulations Over Life Time: Unique Active Superior than Vitamins; SOFW Journal, 134(9) (2008) 52, 54-56, 58, 60. (3) T. Rudolph, J, Pan, R. Scheurich, F. Pfluecker, R. Graf, H. Epstein; Superior two step approach to completely photoprotect avobenzone with a designed organic redox pair; SOFW Journal (2009), 135(9), 14, 16-18. (4) T. Herrling, L. Zastrow, J. Fuchs, N. Groth. Electron spin resonance detection of UVA-induced free radicals; Skin Pharmacol. Physiol. 17 (2002) 381 383 (5) M. Stücker, C. Moll, P. Altmeyer; Sauerstoffversorgung der Haut Unter besonderer Berücksichtigung der kutanen Sauerstoffaufnahme aus der Atmosphäre; Hautarzt 55 (2004) 273-279. (6) T. Herrling, J. Fuchs, J. Rehberg, N. Groth; UV-induced free radicals in the skin detected by ESR spectroscopy ans imaging using nitroxides. Free Radical Biology & Medicine 35 (2003) 59 67 (7) K. Jung, M. Seifert, T. Herrling, J. Fuchs; UVgenerated free radicals in skin: Their prevention by sunscreens and their induction by self-tanning agents. Spectrochimica Acta Part A 69 (2008) 1423 1428 (8) I. Ermakov, J. Lademann, M Ermakova, W. Gellermann; Noninvasive selective detection of lycopene and β-carotene in human skin using Raman spectroscopy; J Biomed Opt, 9(2) (2004) 332-338. (9) F. Ramel et al.; Chemical quenching of singlet oxygen by carotenoids in plants; Plant Physiology, 158(3) (2012) 1267-1278. (10) B. Auffray; Protection against singlet oxygen, the main actor of sebum squalene peroxidation during sun exposure, using Commiphora myrrha essential oil; Int J Cosmet Sci, 29(1) (2007) 23-29. (11) L. Zastrow, N. Groth, F. Klein, D. Kockott, J. Lademann, R. Renneberg, L. Ferrero; The Missing Link Light-Induced (280-1600 nm) Free Radical Formation in Human Skin; Skin Pharmacol Physiol, 22 (2009) 31-44. (12) F. Hyodo. K. Yasukawa, K. Yamada, H. Utsumi; Spatially resolved time-course studies of free radical reactions with an EPRI/MRI fusion technique; Magnetic Reson Med, 56 (2006) 938 943 Acknowledgement The authors thank Dr. Katinka Jung (Gematria Test Lab GmbH) for measurements of the radical status factor (RSF). Authors addresses: *Thomas Rudolph Sylvia Eisenberg Merck KGaA Frankfurter Str. 250 64293 Darmstadt Germany Email: sylvia.eisenberg@merckgroup.com **Julie Grumelard Bernd Herzog BASF Grenzach GmbH Köchlinstr. 1 79639 Grenzach-Wyhlen Germany Email: bernd.herzog@basf.com n 14 SOFW-Journal 140 3-2014