Hydrolyzed collagen interferes with in vitro photoprotective effectiveness of sunscreens

The chronological skin aging is a progressive and natural process with genetic and physiological changes. However, ultraviolet (UV) radiation may accelerate the oxidative stress, generating carcinogenesis and photoaging. Natural compounds and their applications are considered a trend in the cosmetic market. The protein-based film-forming compounds play an important role, once it collaborates for the better distribution of sunscreens on the skin. Here we investigated the in vitro photoprotective effectiveness of sunscreens containing the hydrolyzed collagen associated with UVA, UVB and/or inorganic filters. Sunscreens were developed with octocrylene (7.5%), butyl methoxydibenzoylmethane (avobenzone) (3.0%) and/or titanium dioxide (5.0%), associated or not with the hydrolyzed collagen (3.0%). In vitro photoprotective effectiveness was determined in a Labsphere® UV2000S by the establishment of the sun protection factor (SPF) and critical wavelength (nm) values. Physicochemical and organoleptic characteristics were also assayed. The hydrolyzed collagen subjectively improved the formulation sensory characteristics. However, this bioactive compound led to a decrease of the SPF values of the photoprotective formulations containing octocrylene alone and octocrylene + butyl methoxydibenzoylmethane + TiO2. This inadequate interaction may be considered during the development of new sunscreens intended to contain protein-based components.


INTRODUCTION
Towards the global knowledge about skin cancer and premature aging, public health authorities and experts still consider that the daily use of sunscreens should increase.Additionally, a cumulative exposure to ultraviolet radiation (UVB: 290-320 nm and UVA: 320-400 nm) accelerates photoaging, leading to an imbalance between endogenous oxidants and antioxidant molecules (Bianchi, Antunes, 1999;Hirata, Sato, Santos, 2004;Bagatin, 2009;Rivas et al., 2009;Brasil, 2014).
Brazil is one of the largest producers and exporters of meat, generating many sub-products such as pig skin, bovine hide, bones and others that become natural and sustainable sources of proteins, mainly collagen (Zucchi, Caixeta-Filho, 2010) that is regarded as one of the most useful biomaterials.The excellent biocompatibility and safety due to its biological characteristics, such as biodegradability and weak antigenicity, made collagen a resource for cosmetic use (Nehlyudov, 2003;Teixeira, 2006;Walrand et al., 2008;Gomez-Guillén et al., 2011;Wang, Lim, 2011;Wang et al., 2013;Comblain et al., 2015).
The use of natural and sustainable compounds substances in skin care products has become a trend nowadays.In the past years, the cosmetic market has included the collagen in its formulas as a consequence of its multifunctional characteristics, such as moisturizing (Walrand et al., 2008;Gómez-Guillén et al., 2011;Silva, Penna, 2012).In this context, the properties of collagen can be divided in two categories.First, the properties associated with its gelling behaviour, i.e., gel formation, texturizing, thickening and water binding capacity; and second, the properties related to their surface behaviour, which include emulsion and foam formation and stabilization, adhesion and cohesion, protective colloid function, and film-forming capacity (Bordbar et al., 2013;Wang, Lim, 2011;Wang et al., 2013;Walrand et al., 2008;Zague et al., 2011).
Here we selected two well-established chemical and physical UV filters: butyl methoxydibenzoylmethane (avobenzone), octocrylene and titanium dioxide.Butyl methoxydibenzoylmethane, a potent UVA filter, was the first FDA-approved organic agent to filter UVA radiation.However, it is highly photolabile and after 1 h of sun exposure it decreases its photoprotective properties by half.Additionally, it can affect the stability of other sunscreen ingredients.Besides octocrylene ability to absorb the UVB radiation, it also improves photostability and it is a common choice for sunscreens available at the market.Titanium dioxide is a metallic oxide common used as inorganic UV filter by its higher refractive index, which provides a superior UVB protection and a whiter tone (Kockler et al., 2012;Sambandan, Ratner, 2011).
Oliveira and co-workers (2015), proved through in vivo studies the skin biocompatibility of samples containing butyl methoxydibenzoylmethane 3.0% w/w, associated or not to a natural compound (rutin 0.1% w/w), as well as, Peres and co-workers (2016), determined the cutaneous compatibility of sunscreens containing octocrylene 10.0% w/w, with or without rutin 0.1% w/w.Both in vivo protocols were performed with 17 volunteers and the samples were applied in theirs volar forearms using epicutaneous patches during 24h.Distinct bioengineering approaches were used to assess the biocompatibility and the variables found to be the most representative of skin tolerance were cutaneous redness, hydration and transepidermal water loss (TEWL).Butyl methoxydibenzoylmethane and octocrylene were well-tolerated by the skin, confirming their good biocompatibility.
Here we investigated the photoprotective effectiveness, obtained in vitro, of sunscreens (O/W emulsions) containing the hydrolyzed collagen (3.0% w/w) associated with UVA, UVB organic and inorganic filters.

Development of the formulations
Eight O/W emulsions were developed, containing one or more UV filters, octocrylene (7.5% w/w), butyl methoxydibenzoylmethane (3.0% w/w) and titanium dioxide (5.0% w/w), in the collagen absence or presence (3.0% w/w).Samples were stored for 72 hours prior to physical, physicochemical and in vitro photoprotective efficacy evaluation.Also, two formulations without UV filters were used as controls (F1 and F2).Table I shows the qualitative and quantitative composition % (w/w) of the formulations (FDA, 2007).
Samples were prepared as follows: first, oil phase emollients and the emulsifier were heated with octocrylene and butyl methoxydibenzoylmethane. After, water phase components (including the hydrolyzed collagen) were added, and the mixture was mechanic stirred (1,500 rpm).The water phase was added to the oil phase and the mixture was mechanic stirred (1,500 rpm) for 3min.At the end, titanium dioxide was added into the samples, previously dispersed in isopropyl myristate.
Apparent viscosity and pH values were evaluated in a Fungilab ® , Visco Star R, viscometer, and in a Quimis ® pH meter, respectively.Organoleptic characteristics were analysed subjectively by means of colour, odour and appearance.The results were obtained in triplicate (Anvisa, 2004;Sarruf et al., 2013).

Photoprotection efficacy in vitro
The in vitro photoprotective effectiveness was performed using an UV-2000S Ultraviolet Transmittance Analyzer equipped with an integrating sphere (Labsphere ® , USA).Samples were weighed and uniformly applied with a glove-coated finger on quartz plates with Transpore ® tape (3M, Canada), as the substrate.Samples were then allowed to dry at room temperature for 20 min, protected from light.The analyses were carried out in replicates of three, and nine different points per plate were measured for each sample.The average of the obtained spectral absorbance values were used by the Labsphere ® software UV2000 to calculate in vitro SPF and critical wavelength (nm) values (Velasco et al., 2008;Diffey et al., 2000;Cosmetics Europe, 2011;Wang, Lim, 2011;Oliveira et al., 2016).

Statistical analysis
Statistical analysis was performed by the software Minitab ® (Version 16), with a significance level of 5% (a = 0.05).Data were statistically treated using oneway ANOVA followed by the Tukey test for multiple comparisons and paired t-test.Regarding the formulations, they were divided into pairs according to the presence or absence of collagen in association with the UV filters.The p-values between each group were calculated by the two-sample t-test for equal means, with a confidence interval of 95%.

RESULTS AND DISCUSSION
Most sunscreens perform their efficacy through a combined chemical and physical UV protection and they should meet particular requirements like, for example, balanced UVA/UVB efficacy with high level of protection, water resistance, easy of application with film-forming property, uniform protection, high stability, and good skin biocompatibility (Balogh et al., 2011;Stiefel, Schwack, 2014;Oliveira et al., 2016).
Formulations showed satisfactory stability with high viscosity (14400 to 33500 cP), possibly due to the water retention capacity of the medium, in addition to the emulsifying and stabilizing properties of hydrolyzed collagen.The systems had pH values biocompatible with skin (4.84 to 7.71) (Walrand et al., 2008;Gómez-Guillén et al., 2011;Silva, Penna, 2012).Stratum corneum (SC) presents an acid mantle that contributes with the skin acidic surface, which is relevant for good tissue condition and homeostasis, considering the skin microflora control and support of physiological processes (Lambers et al., 2006).The activities of the skin enzymes, for instance, are significantly reduced with the increasing of the SC pH.Likewise, SC proteases are more efficient at near-neutral or alkaline pH.Several skin disorders are consequences of disturbed enzyme activity, for example, Gaucher's disease is due to altering β-glucocerebrosidase activity, and a deficiency in acidic sphingomyelinase cases the Niemann-Pick disease (Natarajan et al., 2014).According to this scenario, a topical product projected to be applied in high extensions of the cutaneous tissue must have pH value compatible with the skin.
The presence of hydrolyzed collagen improved the organoleptic characteristics of the developed samples, subjectively, increasing their brightness and spreadability.In respect to the colour, a slight acceptable modification was observed, probably due to the dark yellow characteristic coloration of the incorporated active compounds.
F1 and F2 samples did not absorb the radiation with, approximately, 100% transmittance at the UVA and UVB regions, since they were UV filters-free.Regarding the in vitro photoprotective effectiveness, sunscreens containing the UVA filter (butyl methoxydibenzoylmethane) (F5 and F6), TiO 2 (F7 and F8) and octocrylene + UVA filter + TiO 2 (F9 and F10), containing or not the hydrolyzed collagen, developed indicative profiles of broad-spectrum action against UV radiation, with critical wavelength over 378 nm (Dominique, 2008;Rai, Shanmuga, Srinivas, 2012).This type of broad-spectrum absorption profile is of utmost importance for sun-care products, considering the harm  affects from radiation.Excess of UV radiation causes damages to the skin and hair, such as sunburn, photoaging, local dryness, and cancer, as well as, immunosuppression.UVB (mainly) and UVA may provoke direct cellular DNA harm, triggering an inflammatory response and melanin production.It has been confirmed by an explorative intraindividual study that a broad-spectrum sunscreen with a high protection factor is able to prevent UV-induced erythema and hyperpigmentation in health volunteers (Khun et al., 2016).Actually, the formulation containing all the three UV filters and hydrolyzed collagen showed an UVA/UVB rate of 0.92 (data not shown), considered of high UVA protection (Baby et al., 2009).
The UVB protection was more evidently achieved with the formulations containing the octocrylene, with SPF values above 10 (Kaimal, Abraham, 2011), although, the combination of hydrolyzed collagen and octocrylene decreased the SPF in 41% (F3 and F4), while the association of the three UV filters with the hydrolyzed collagen led to a SPF decay of 38% (F9 and F10).These results showed that this natural component contributed inadequately to the absorption on the UV range established in vitro.Table II shows the in vitro photoprotective effectiveness of the sunscreens.
Interactions among actives, bioactives from natural sources, excipients and vehicles of sunscreens may generate negative responses that compromise the efficacy of these systems, like active compound degradation with the respective formation of reactive species of oxygen.Also, studies on photochemical behaviour of UV filters are an important part of sunscreen development.Through interactions with sunlight, artificial light or several substances used to obtain the final product, it may provoke the photodegradation of UV filters, resulting in decreased protection ability and formation of new by-products with possibly different properties (Kockler et al., 2014;Stiefel, Schwack, 2014).
Considering the use of protein-based bioactives, Graziola and co-workers (2016), observed that gelatin at 5.0% w/w, as an amorphous raw-material, was able to improve the SPF of mineral oil-based dispersions containing 6.0% w/w of benzophenone-3 or 7.5% of ethylhexyl methoxycinnamate, from 2.8 to 4.1 and 6.4 to 8.4, respectively.The critical wavelength (nm) of those dispersions was not affected, being kept below 354.5 nm, even in the UVA filter (benzophenone-3) presence (Graziola et al., 2016).Stiefel and Schwack (2014), identified UV spectra alterations of the octocrylene incubated at 37 °C with bovine serum albumin (BSA) compared to the UVB filter alone.The interaction between octocrylene and BSA, after incubation, was assumed to be a result of covalent bound of the UVB filter to BSA through Michael addition followed by ethylhexyl cyanoacetate elimination (Stiefel, Schwack, 2014).This reaction has led to a maximum wavelength displacement from 310 to 280 nm, approximately, and a reduction in absorbance intensity.Stiefel and Schwack (2014) also detected interactions between octocrylene and gelatin after simulated irradiation.These behaviours could possibly impact the SPF value, regarding its parameter property of defence mainly against UVB radiation, as observed in our research, when the SPF values of the samples containing octocrylene associated with the hydrolyzed collagen have diminished.

CONCLUSION
The hydrolyzed collagen moisturizing and texturizing properties subjectively improved the formulation sensory characteristics.However, this bioactive compound at 3.0% w/w did not influence the critical wavelength (nm) of the sunscreens and it led to a decrease of the SPF values, especially, of the high UVB photoprotective formulations containing octocrylene at 7.5% w/w (alone) and octocrylene (7.5% w/w) + butyl methoxydibenzoylmethane (3.0% w/w) + TiO 2 (5.0%w/w).This identified interaction, according to the in vitro established efficacy, may be considered by the Research & Development professionals during the elaboration of new sunscreen products intended to contain protein-based components.

TABLE I -
Qualitative and quantitative composition (% w/w) of the O/W formulations

TABLE II -
Sunscreens in vitro photoprotective effectiveness Legend: N.A.: not applicable.Values reported as mean ± standard deviation.The p-values between each group were calculated by the two sample t-test for equal means, with a confidence interval of 95%.