The need for appropriate UV protection of the skin against the damaging effects of UV radiation is well recognized. While UVB filters are already widely used also in daily care products, adequate and long-lasting UVA protection seems still underrepresented in many sun care and especially in daily care formulations, although UVA light is known to be particularly responsible for long-term effects like premature skin-aging (photoaging), and also for DNA damage, suppression of the immune system, skin cancer and other deleterious effects.
Keywords
Diethylamino hydroxybenzoyl hexyl benzoate (DHHB), UVA protection, UVA radiation, free radicals, reactive oxygen species, UVA balance, Integrated Sun Protection Factor (ISPF), photoaging, electron spin resonance spectroscopy, ethylhexyl triazone, ethylhexyl methoxycinnamate, zinc oxide (and) triethoxycaprylylsilane, sunscreen.
Summary
UVA rays can cause severe damage within the epidermis and the dermis via the generation of aggressive free radicals, the so-called ‘reactive oxygen species’ (ROS), which attack targets inside and outside the cells, leading to, for example, lipid peroxidation, oxidative DNA damage, and enhanced matrix protein degradation.
In the present study the protective effect of diethylamino hydroxybenzoyl hexyl benzoate (DHHB), an efficient and photostable UVA-I filter against the generation of free radicals, has been investigated in human skin biopsies by quantitative electron spin resonance (ESR) spectroscopy after full-spectrum solar simulated irradiation. Radical stabilisation was achieved with the spin trap PBN (phenyl-tertbutylnitrone). The radical protection performance of the formulation has been expressed using so-called ‘integrated sun protection factor’ (ISPF). We compared the results of DHHB with those obtained from different types of UV filters – ethylhexyl methoxycinnamate (OMC), coated zinc oxide, and a commercially available mixture of DHHB and OMC. Incorporated into an O/W emulsion, DHHB revealed an exceptional high radical protection and showed a good dose-effect relationship. Coated zinc oxide yielded moderate activity, whereas UVB filters like OMC achieved only a low performance against free radicals. This is reasonable as 90% of free radicals are induced by UVA light.
The results of this study show that DHHB is able to reduce significantly the number of UV-induced free radicals in skin even when applied at low levels, thus providing high performance protection against the major source of premature skin aging – free radicals. Due to its exceptional performance combined with an excellent photostability and a high formulation flexibility, DHHB represents an indispensable tool in the daily battle against UVA-induced skin damage.
Introduction
UV radiation is well recognized for exerting detrimental effects on human skin. The trend towards higher sun protection factors (SPF) may protect our skin from overdoses of UVB light, and thus provide protection from the most directly perceivable and painful effect of sunburn, but also from DNA damage, suppressed immune reaction and finally from photocarcinogenesis. However, even high SPF values are not necessarily connected with appropriate protection from UVA light. Their high protection numbers seduce people into prolonged and careless sun exposure. This may lead in the worst cases to more or less unfiltered (over)exposure to UVA radiation without any warning on the skin, as an overdose of UVA radiation is not directly and immediately perceivable.
This deficiency has now been addressed by the European Commission Recommendation on the efficacy of sunscreen products and the claims made relating to them1. For sunscreen products a minimum UVA protection is required. It should be closely related to the level of UVB protection of the product. More precisely, the level of UVA protection, measured in the persistent pigment darkening test (or an adequate alternative in vitro test), should be at least 1/3 of the SPF in vivo value, the latter addressing mainly UVB radiation.
However, up to now there is no comparable recommendation for the daily protection of the skin, although the need for regular, reliable and adequate protection of the skin against the long-term effects of UVA protection is scientifically well documented and has been commonly recognized for a long time.
The result of the cumulative effects of even sub-acute UVA doses on human skin is clearly visible, but after years as premature skin aging, or photoaging, increased wrinkling, loss of skin elasticity and skin moisture, pigment disorders, and thickened and leathery skin. On a biochemical level the induction of stress genes, up-regulation of matrix-metalloproteinases (MMPs), giving rise to enhanced elastin and collagen degradation, peroxidation of (membrane) lipids and other intra- and extracellular components, induction of inflammatory reactions, oxidative DNA damage, and also immunosuppressive effects are well-described consequences of UVA radiation.
Ninety-five per cent of the solar UV spectrum reaching the Earth’s surface is accounted for by UVA light, and only 5% by UVB light. UVA radiation penetrates much deeper into the skin than UVB light, and it is not even blocked by window panes. Nonetheless, adequate, reliable and well-balanced UVA protection seems still underrepresented especially in daily care formulations, where increasing UVB protection factors attain levels (SPF 15 and higher) comparable to those offered on typical sunscreen products for the trip to the beach, but where UVA protection labelling is still unusual.
UVB light is able to damage skin constituents by direct photochemical interaction with UVB absorbing molecules in the uppermost skin layers, inducing for example photodimerisation of thymidine bases in the DNA. UVA radiation, on the other hand, acts predominantly indirectly through the generation of aggressive free radicals after primary sensitization of skin chromophores. The so-called reactive oxygen species (ROS) – e.g. hydroxyl radicals, superoxide radicals, lipid radicals – attack targets within and outside of the cells, leading to the detrimental effects mentioned above (Figure 1).
These findings show that UVA radiation represents a steady but unperceived risk and that efficient UVA protection on a daily basis is necessary to prevent long-term consequences caused by regular sub-acute UVA exposure. Hence, an efficient and photostable UVA filter should provide the highest efficiency in reducing the number of generated free radicals, the origin of skin damage mentioned above. The power of free radical protection can be expressed by a new radical protection factor, the so-called Integrated Sun Protection Factor (ISPF). It is defined in a similar way to the traditional SPF, as the ratio of UV radiation energy needed to generate a certain number of free radicals in a defined skin volume (= biological endpoint) with sunscreen protection compared to the energy needed to generate the same number of free radicals in the skin without UV protection2, 3. This radical protection factor covers effects derived from UVA and UVB radiation in all skin compartments down to the subcutis.
The study presented here targeted the exceptional performance in radical protection of the modern photostable UVA-I absorber DHHB (Figure 2) and to compare its efficacy with other types of UV filters and UV filter combinations.
Material and methods
Filter platforms were tested in a standard O/W formulation base (Table 1).
Abbreviations used: SPF (sun protection factor), ISPF (Integrated Sun Protection Factor), DHHB (diethylamino hydroxybenzoyl hexyl benzoate), EHT (ethylhexyl triazone), OMC (ethylhexyl methoxycinnamate), coated zinc oxide (zinc oxide (and) triethoxycaprylylsilane), iAMC (iso-amyl p-methoxycinnamate), BMDBM (butyl dimethoxydibenzoylmethane), MBBT (methylene bis-benzotriazolyl tetramethylbutylphenol), BEMT (bis-ethylhexyloxyphenol methoxyphenyl triazine).
The materials and methods used in the present study for the quantification of free radicals in skin biopsies have already been described in detail elsewhere2, 3.
The principles of the method
1. Human skin biopsies are incubated with a spin trap (400 mM phenyl-tert-butylnitrone, PBN).
2. After rinsing and drying the biopsies are exposed to UV irradiation either blank or covered by a 2 mg/cm2 sunscreen formulation layer (Atlas CPS+ Suntest, 9.53 mW/cm2, UVA+UVB), irradiation times: 150 s, 300 s and 600 s.
3. Directly after UV exposure the sample is subjected in the same sample holder to quantification of free radicals by ESR spectroscopy (ERS 300+, ZWG Germany, modified by Galenus; spectrometer settings see 2, 3 (Figure 3).
4. The quantification of the generated free radical–PBN adducts is performed by peak to peak signal amplitude. The signal amplitude is compared to the amplitude of a calibration probe (standard) with a known number of free radicals. This standardisation is the basis for the calculation of the RG value (RG = Radicals Generated). The RG value describes the number of trapped free radicals/ROS and is presented as a real number X multiplied by 1012 and normalized to 1 mg of the investigated probe (human skin biopsy).
5. The calculation of the ISPF is similar to that of the erythemal SPF: the ratio of UV doses (E) required to reach the same endpoint in protected skin (Ep) and unprotected skin (Eu): the same number of free radicals (RG).
ISPF = Ep / Eu (1)
6. This calculation is possible because the number of free radicals generated (RG = X x 1012 radicals/mg skin) correlates well with the UV dose supplied and is independent of the exposure time. The following equation describes the dependency of the free radicals induced on the energy applied. It achieves a close correlation with the experimental data, according to a simple numerical adjustment of parameters a and b through the least-square error method.
log (1000 x X) = a x log E + b (2)
X = number of free radicals generated, E = UV dose (mJ/cm2)
Transmittance spectroscopy was performed using an Optometrics 290 S, roughened PMMA plates (Schönberg GmbH, Germany) were used as substrate.
Results and discussion
With a maximum absorption of DHHB at 354 nm and an A1/1 value of 910-940, this filter represents a strong long-wave UVA-I absorber. Figure 4 shows the absorption spectrum of DHHB as well as a spectrum of a commercially available liquid mixture of 35% DHHB in OMC, the latter representing an easy to use broad-spectrum solution matching easily the UVB to UVA ratio of 3:1 required by the European Commission Recommendation1.
Due to an intramolecular rearrangement (H-Shift), the molecule is able to transform the excitation energy into heat without photochemical degradation – thus promising excellent photostability and reliable protection from UVA rays and thus from UV-induced generation of free radicals in the skin, and consequential from skin damage (Figure 5). A series of different sun filters and filter combinations (Table 1) have been tested for their performance against UV-induced free radicals by quantitative ESR spectroscopy on human skin biopsies.
Figure 6 shows the dependency of the number of free radicals generated in the skin biopsy from the UV dose applied (9.53 mW/cm2 UVA+UVB), as well as the significantly varying protection efficiencies of the different UV filter types tested.
The formulation base itself (without UV filters) already shows a small effect in decreasing the number of free radicals compared to unprotected skin, and also the pure UVB filter OMC (6.5%), providing a reasonable UVB protection of SPF 9, yields only poor protection with a view to free radical generation. The low, but very broadly absorbing inorganic pigment (3.5% coated zinc oxide) shows a moderate performance. The pure organic UVA absorber DHHB (3.5%) on the other hand exhibits a significantly higher efficacy in reducing the number of free radicals generated upon UV irradiation. The broad-band combination of DHHB (3.5 %) with the UVB absorber OMC (6.5%) gives a further increase in radical protection and, of course, additional protection against sunburn (SPF 14). In Figure 7 the number of free radicals generated in a protected skin biopsy upon a certain irradiation dose is considered in relation to the number of free radicals generated in the filter-free formulation base (=100%).
Ethylhexyl methoxycinnamate (OMC, 6.5%), a sunscreen that is still frequently used in daily care products as the only UV-shielding system, is not able to reduce much more than 1/10 of the free radicals generated, whereas 3.5% of coated zinc oxide, a broad-band filter often regarded to give only poor efficiency, provides a 30% radical protection. Diethylamino hydroxybenzoyl hexyl benzoate, being a strong and photostable UVA-I filter, attains a more than 50% reduction of full-spectrum UV-induced free radicals. Furthermore, independently from the dose applied (150–300-600 s, 9.53 mW/cm2), the relative protection performance of respective filtering system remains at the same level.
In Figure 8 the ISPF and the SPF values are compared for different filter types and filter combinations. Looking at the ratio between radical and erythemal protection factors it is clear that a high SPF is not necessarily connected with appropriate UVA protection.
For OMC (6.5%) the discrepancy of the protection factors (SPF 8.9/ ISPF 1.64 = 5.4) is by far the highest, the pure UVB filter providing scarcely more protection against free radicals than the filter-free formulation. The SPF, up to now often the only protection index labeled on a sunscreen product and mostly on daily care products, may be a false indication of the level of protection for the end consumer, which seduces them into prolonged or careless sun exposure without providing significant protection against deep penetrating UVA rays. The low, but very broad absorbing zinc oxide, on the other hand, gives a much better ratio, but still at a low level (SPF 7.2/ ISPF 2.49 = 2.9). By far the best and most balanced ratio between erythema prevention and radical protection we found was with pure DHHB. Both protection factors are in the same order of magnitude (SPF 4.9/ISPF 6.6 = 0.7), providing well-balanced protection. The risk of getting sunburned every day is rather low, however the long-term exposure of our skin to UVA radiation – which can even happen through glass – means that the protection requirements for a daily care product should provide at least the same or even higher levels of protection against UVA radiation as against sunburn. With these thoughts as a principle basis, DHHB (3.5%) represents a perfectly balanced UV protection with a five-fold protection against sunburn, which is, according to Rudolph et al4, more than sufficient for regular daily care protection in mid-European conditions, combined with a more than six-fold protection against free radicals and thus against the long-term effects of UVA light – i.e. premature skin aging. However, the final scientific evidence about the appropriate level of UVA or radical protection for daily care is still not available.
The broad-band combination of DHHB (3.5%) and OMC (6.5%) revealed a ratio of erythemal and radical protection of SPF 14/ ISPF 8.32 = 1.7. This is more suitable for typical sunscreen application, where the short time exposure to higher irradiation doses, for example during sport, a trip to the beach and other outdoor activities that carry the acute risk of sunburn of untrained skin. Here, the required minimum protection level against long-term UVA- induced damage is important and should be closely related to the level of UVB protection, but less prominent. This specific requirement for sunscreen products is now well mirrored in the European Commission Recommendation1.
The level of free radical protection desired in a formulation with a given UVB protection can be easily adjusted with the level of DHHB. In our study we have observed a good correlation of the radical protection factor ISPF and the percentage of DHHB used (Figure 9) in formulations with a constant level of the UVB filter EHT. Furthermore, we compared radical protection performance of DHHB alone and in combination with other UVA and broad-band systems (Figure 10). Due to its high performance combined with an excellent photostability, DHHB (3.5%) showed a similar to higher performance in SPF and ISPF values than BMDBM (5%)1. To reveal the broad-band protection performance of the filter combination DHHB–EHT in comparison to other broad-band filter platforms Figure 10 shows the corresponding SPFs and ISPFs of a formulation containing DHHB (7%) combined with 2% EHT versus a mixture of BMDBM (3%) + BEMT (2%) + MBBT (8%, 4 % active) + iAMC (5%)2. Both filter platforms revealed similar results and both platforms provided a very good protection performance in the UVB with SPFs 19 and 21, respectively, as well as against free radicals with an ISPF 12, however at different levels of total filter used (9% versus 14%).
Conclusion
For an adequate and well-balanced daily protection of the skin from the detrimental effects of UV rays, efficient prevention from the generation of free radicals, and thus especially from UVA-light, is necessary. The new UVA-I filter DHHB has been shown to provide excellent performance in radical protection – thus representing an efficient and indispensable tool in the daily battle against UVA-induced damage of human skin.
References
1. European Commission Recommendation on the efficacy of sunscreen products and the claims made relating thereto. Official Journal of the European Union (2006/647/EC).
2. L. Zastrow et al, Integrated sun protection factor: A new sun protection factor based on free radicals generated by UV irradiation. Skin Pharmacol. Physiol., 2004, 17, 219-231.
3. L. Zastrow et al., A major innovation in sun protection measurement – The integrated sun protection factor ISPF. SOEFW-Journal, 2006, 132, 12-19.
4. T. Rudolph et al, Using real life monitoring to design a daily skin care product, 22nd IFSCC Congress. Edinburgh, 2002, Podium 23.
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