Protective effect of a hydrogel containing Achyrocline satureioides extract-loaded nanoemulsion against UV-induced skin damage

Journal of Photochemistry & Photobiology, B: Biology 163 (2016) 269–276

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Protective effect of a hydrogel containing Achyrocline satureioides extract-loaded nanoemulsion against UV-induced skin damage L.A. Balestrin a, J. Bidone a, R.C. Bortolin b, K. Moresco b, J.C. Moreira b, H.F. Teixeira a,⁎ a b

Programa de Pós-graduação em Ciências Farmacêuticas da Universidade Federal do Rio Grande do Sul, Porto Alegre, RS 90610-000, Brazil Departamento de Bioquímica da Universidade Federal do Rio Grande do Sul, Porto Alegre, RS 90035-003, Brazil

a r t i c l e

i n f o

Article history: Received 3 December 2015 Accepted 24 August 2016 Available online 26 August 2016

a b s t r a c t Achyrocline satureioides is a medicinal plant widely used in South America that exhibits a well-documented antioxidant activity. Such activity has been related to their main aglycone flavonoids quercetin, luteolin, and 3-Omethylquercetin (3MQ). This study addresses the development of antioxidant hydrogels containing an A. satureioides extract-loaded nanoemulsions aimed at topical application. The systems investigated were A. satureioides extract-loaded nanoemulsions (ASNE) obtained by spontaneous emulsification procedure formulated in semisolid hydrogels composed of Carbopol® Ultrez 20 (HASNE). Hydrogels exhibit a non-Newtonian pseudoplastic behavior. A higher release of 3MQ from ASNE (3.61 μg/cm2/h) was observed when compared with HASNE (2.83 μg/cm2/h). Different parameters that may have an influence on the retention of flavonoids into the skin were investigated by using a Franz-type diffusion cells. Indeed, the amount of formulation applied on donor compartment was found to play a crucial role. At the optimized conditions, retention of approximately 2 μg/cm2 of flavonoids was detected into the skin. A higher retention of 3MQ was detected (approximately 1.0 μg/ cm2) in comparison with the other flavonoids. Finally, a protection the porcine ear skin by formulations, against oxidative stress generated by UVA/UVB light was demonstrated by means of TBARS, protein carbonylation, and protein thiol content assays. The overall results showed the potential of the formulations developed in this study for the prevention of oxidative stress on the skin. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Overexposure to ultraviolet radiation is considered as one of the main causes of skin aging and carcinogenesis [1,2]. Free radicals generated by sunlight on the skin are able to oxidize biomolecules, causing disorders at different levels [2,3]. Natural enzymatic and non-enzymatic antioxidants can act as protectors against the damage caused by free radicals. However, high incidence of sunlight and other factors causing oxidative stress, such as pollution and smoking, can overload the body's natural defense system [2,4]. In this way, topical administration of antioxidant flavonoids has been investigated as a strategy to protect the skin from oxidative stress [5,6]. Achyrocline satureioides is a medicinal plant native of the Southeastern region of South America that have been investigated due to its antioxidant activity [7]. Early studies have shown the free radical scavenging and antioxidant activity of extracts of this medicinal plant by evaluating the total antioxidant potential (TRAP) and the inhibition of lipid peroxidation by TBARS (thiobarbituric acid reactive substances) method [8,9]. The protection of rabbit skin against ultraviolet radiation ⁎ Corresponding author at: Avenida Ipiranga, 2752, ZIP Code: 90610-000 Porto Alegre, RS, Brazil. E-mail address: [email protected] (H.F. Teixeira).

http://dx.doi.org/10.1016/j.jphotobiol.2016.08.039 1011-1344/© 2016 Elsevier B.V. All rights reserved.

by the A. satureioides extract incorporated in a preformed oil-in-water emulsion was also demonstrated [10]. Overall, the results showed that the damage was much lower in the treated rabbit skin in comparison with untreated skin, remaining similar to the group of animals that was not irradiated. The antioxidant activity of A. satureioides extracts has been very often related with the high flavonoids content (i.e. quercetin, luteolin, and 3-O-methylquercetin) once a strong antioxidant activity of these compounds is well-documented especially for quercetin [9,10,11,12]. A key consideration in the design of formulations for poorly soluble antioxidant compounds, such as flavonoid aglycones, is their ability to improve the penetration of the drugs/bioactive compounds into the skin layers to provide an adequate protection [4]. In this way, the incorporation of the main flavonoids from crude ethanol A. satureioides extracts into nanoemulsions obtained by means of spontaneous emulsification procedure was described by our research group [13]. Such a procedure proved to be able to incorporate high amount of flavonoids in monodisperse nanoemulsions (200 nm range) and improve their retention into porcine ear skin epidermis, especially when tissues were previously injured [13,14]. More recently, the antioxidant activity of these nanoemulsions was preliminarily evaluated by measuring the inhibition of lipid peroxidation (TBARS) induced by AAPH (2,20azobis-2-ami-dinopropane-dihydrochloride) in a porcine ear skin

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homogenate [15]. An improved protection of skin against lipoperoxidation was observed for the A. satureioides extract-loaded nanoemulsion once a significant lower formation of reactive species was detected in comparison with skin treated with a formulation containing pure quercetin or non-treated skin. Despite these promising results, nanoemulsions are low viscous dispersions and their incorporation in a convenient semisolid pharmaceutical dosage form with proper viscosity for topical application has yet to be performed. Thus, this study described the physicochemical, release, and rheological properties of A. satureioides extract-loaded nanoemulsions incorporated in a Carbopol® Ultrez 20-based hydrogel. The retention of the flavonoid's extract from these formulations into the porcine ear skin and the protection of this tissue against oxidative damage generated by its exposure to UVA/UVB light was also investigated. 2. Experimental 2.1. Chemical and Reagents Egg lecithin (lipoid E-80) and Medium Chain triglycerides (MCT) were acquired from Lipoid (Germany); Polysorbate 80 was supplied from Vetec (Brazil); Vitamin E was purchased from Alpha Chemical (Brazil); Methanol was obtained from JT Barker (USA); Acetonitrile was obtained from Tedia (Brazil); Phosphoric acid was acquired from Merck (Brazil). CabopolÒ Ultrez 20 was kindly donated by lubrizol (Cleveland, USA). 2.2. Flavonoid Assay by HPLC/UV The simultaneous determination of quercetin (QCT), luteolin (LUT) and 3-O-methylquercetin (3MQ) in A. satureioides extracts, formulations and porcine ear skin samples was carried out by using a previously validated HPLC/UV method [13]. The system consisted of a Shimadzu Chromatograph, equipped with LC-10 CE pump, CBM-10 system controller and SPD-20 A detector UV/Vis (362 nm). A Synergi Polar-RP 4.6 × 150 nm id, 4 μm (Phenomenex, USA) column protected by a stainless steel pre-column packed with C18 silica, 150 μm, 140 Å (Phenomenex, USA) was used. The temperature was controlled at 30 ± 1 °C and the mobile phase consisted of methanol:0.16 M phosphoric acid:acetonitrile (46:44:10 v/v/v). The flow of the mobile phase was 0.8 mL/min and the injection volume was 20 μL. The samples were diluted in methanol:16 mM phosphoric acid (50:50, v/v) before analyses. 2.3. Preparation of A. satureioides Ethanolic Extract Inflorescences of A. satureioides were acquired from Chemical, Biological and Agricultural Pluridisciplinary Research Center of Campinas State University/CPQBA-UNICAMP (São Paulo, Brazil). A voucher specimen was deposited in the herbarium of CPQBA-UNICAMP under number 308. The extract of A. satureioides (ASNE) was prepared from inflorescences by maceration using ethanol 80% (v/v) for 8 days. A proportion of 7.5% (v/v) of plant was used in the extraction. The extract was pressed and filtered before the use.

nanoemulsion. The pH was adjusted to 7.0 with 0.5 M NaOH solution. A control formulation containing blank nanoemulsions was prepared in similar conditions (HNE). 2.5. Characterization of Formulations Nanoemulsions were characterized before and after their incorporation into hydrogel by the determination of mean droplet size, polydispersity index, ζ-potential, droplet morphology, as well as flavonoids content. The droplet size and polydispersity index were determined by photon correlation spectroscopy (PCS) at 25 °C after dilution of the samples in purified water. ζ-potential was determined by electrophoretic mobility after diluting the samples with 1 mM NaCl solution. The measurements were performed using a Zetasizer Nano-ZS90® (Malvern Instruments, England) equipment. The droplet morphology of the nanoemulsions was evaluated by transmission electron microscopy (TEM). Previously, the samples were diluted in water (1:10), distributed on formvar-coated copper grid (200 mesh) and stained with uranyl acetate (2%). The images were obtained using microscope JEM1220EXII (Jeol Ltd., Akishima, Japan). For flavonoid content determination, the samples were diluted in methanol and methanol:16 mM phosphoric acid (50:50, v/v) before HPLC analyses. These physicochemical properties were also evaluated after 90 days of storage at 4 °C. The reological evaluation of the HASNE was carried out using a Brookfield RVDV II+ viscosimeter and spindle No 21. The results were presented as the relation between shear stress (D/cm2) and shear rate (s− 1). Moreover, the data were fitted in different rheological flow models, as Binghan, Ostwald, Casson, and Herchel-Bulkley to predict flow behavior. 2.6. Skin Permeation/Retention of Flavonoids QCT, LUT and 3MQ permeation/retention from formulations was evaluated using Franz-type diffusion cells (DIST, Brazil). Porcine ears were gently donated by Ouro do Sul-Cooperativa dos Suinocultores do Caí Superior Ltda (Harmonia, Brazil). The ears hair was cut and the skin was removed from cartilage using scissors and scalpel. After that, the skin was cut in circular pieces and stored at − 20 °C until use. On the day of experiment, the pieces were rehydrated with Phosphate Buffer (PBS) pH 7.4 for 30 min and placed between the donor and receptor compartment of the Franz diffusion cell (2.54 cm2 area). Aliquots of nanoemulsion ASNE (10, 30, 50, 100, 250, 500, and 1000 μL) or hydrogel HASNE (100 μL) were placed directly on the skin into donor compartment. Then, the receiver compartment was filled with receptor fluid (pH 7.4 phosphate buffer: ethanol, 70:30 v/v). The temperature (32 ± 1 °C) and stirring were controlled throughout the experiment. After 8 h, an aliquot of receptor fluid was collected and the skin removed from the apparatus. The skin was cleaned with cotton, tape stripped with one adhesive tape (3 M Scoth®), and cut in small slices. Then, the flavonoids were extracted with methanol in ultrasound bath for 30 min and quantified by HPLC. In turn, the receptor fluid aliquots were diluted with methanol:16 mM phosphoric acid (50:50, v/v) and analyzed by HPLC. 2.7. Evaluation of 3MQ Release from HASNE

2.4. Preparation of Formulations Nanoemulsions were prepared by spontaneous emulsification procedure as previously described [16]. The components of the oil phase (egg-lecithin, MCT, vitamin E and extract) were solubilized in ethanol and poured into the aqueous phase (water and polysorbate 80) under constant stirring. Then, the solvent excess was removed under reduced pressure until the desired volume. The final liquid formulation contained 1% of dry residue of A. satureioides extract (ASNE). In turn, the hydrogels containing nanoemulsions (HASNE) were obtained by adding 0.15% of the gelling agent (Carbopol® Ultrez 20) into

3MQ release from ASNE and HASNE through cellulose esters membranes (50 nm pore diameter, Milipore®) was evaluated by using Franz-type diffusion cells (DIST, Brazil). The membranes were hydrated with PBS (pH 7.4) and placed between the donor and receptor compartments. The temperature was adjusted at 32 ± 1 °C and the system was kept under constant stirring. Then, the receptor compartment was filled with PBS (pH 7.4):ethanol (70:30 v/v). An aliquot of the formulations (100 μL of ASNE or 100 μg of HASNE) was placed on the donor compartment and aliquots of the receptor fluid were collected after 0.5, 1, 2, 3, 4, 5, 6, 7 and 8 h, with subsequent replacement with fresh medium. The

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3MQ quantification was performed by HPLC as described in the previous section. 2.8. Antioxidant Activity Studies 2.8.1. Evaluation of the Antioxidant Potential of the Formulations The antioxidant potential of HNE and HASNE was evaluated by means of total reactive antioxidant potential (TRAP) and total antioxidant reactivity (TAR) assays [17]. A solution of the free radical AAPH (2,2-azobis (2-amidinopropane) hydrochloride) with luminol was prepared and kept standing for 2 h at room temperature (22 °C) for stabilization. Then, 180 μL of this solution was added to 20 μL of sample. After, the luminol chemiluminescence intensity induced by AAPH was evaluated in a liquid scintillator counter (Wallac 1450 Micobeta® TriLux, Perkin-Elmer, Boston, MA, USA). TRAP was determined by calculating the area under the curve (AUC) of the luminol chemiluminescence intensity over time, using GraphPad software (San Diego, CA, USA). In turn, TAR was determined as the ratio between the luminol chemiluminescence intensity without the presence of formulation (I0) and the light intensity in the presence of the formulations. Trolox at 40 μM was used as positive control of the experiment. 2.8.2. Evaluation of the Skin Protective Capacity of Formulations The skin protective capacity of HNE and HASNE were evaluated through the determination of Proteins Carbonyls, Total Thiols, and Thiobarbituric Acid Reactive Substances. The porcine ear skin was cut in pieces of 2.54 cm2, and treated with 100 μL of HASNE or HNE for 8 h, as described in the permeation/retention study. After this period, the skin pieces were placed on plates containing filter paper and cotton soaked in phosphate buffer (pH 7.4) to maintain skin moisturize. Then, the plates were disposed in a chamber and exposed to UVA/UVB radiation for 3 h with an UVA lamp Exo Terra Sun. Glo Neodymium 100 W (41 mW/cm2 dose; wavelength 365 nm) and an UVB lamp Exo Terra Repti Glo 15 W (1.8 mW/cm2 dose; wavelength 290 nm). Then samples were washed, homogenized in Ultra-Turrax® with phosphate buffer, centrifuged and the supernatant was analyzed for different parameters. The skin treated was compared with two controls: skin that was not exposed to radiation (NIS) and skin that was not treated with formulations before exposure to the radiation (IS). All results were normalized in terms of total protein content [18]. The assay for determining protein carbonyls was performed as described by Levine et al. [19]. Protein carbonyls of skin homogenates were precipitated by the addition of 20% trichloroacetic acid (in a rate of 1:1 v/v). After centrifugation, the pellet formed was resuspended in 100 μL of 2 M NaOH, being added 100 μL of 10 mM DNPH (dinitrophenylhidrazine). The sample was centrifuged over again; the pellet was washed three times with ethanol/ethyl acetate (1:1 v/v) and resuspended with a solution of 8 M urea (pH 2.3). Finally, the absorbance was obtained by a spectrophotometer at 370 nm. In total tiol assay, the reaction of 5,5-dithiobis-2-nitrobenzoic acid (DTNB) with SH groups, proteic and non-proteics, originates a yellow compound which can be quantified spectrophotometrically. The reaction was conducted as described by Ellman [20]. So, an aliquot of each sample was diluted in PBS (pH 7.4) and mixed to the 10 uL of ethanolic DTNB solution. After 60 min of reaction, the color intensity was measured spectrophotometrically at 412 nm to determine the value of Total Thiol (T-SH). The lipoperoxidation was determined by assessing the level of Thiobarbituric Acid Reactive Substances Assay (TBARS) in the skin according the methodology described by Draper and Hadley [21]. Firstly, skin homogenates were mixed to the 10% of trichloroacetic acid and centrifuged at 10,000 rpm for 10 min. Then, an aliquot of 100 ul of the supernatant was removed, mixed with 100 μL of 0.67% thiobarbituric acid and heated in dryblock for 40 min. The color developed by TBARS presence was determined by spectrophotometer at 532 nm.

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2.9. Statistical Analysis Statistical analysis of the data was performed using analysis of variance (ANOVA) followed by Tukey test, using the 8 ORIGIN software. The differences were considered statistically significant at p b 0.05. 3. Results 3.1. Physicochemical Characterization of Formulations ASNE exhibits droplet size of 250 nm, polydispersity index lower than 0.2, and ζ-potential of -50 mV (Table 1). The thickening of the nanoemulsion with Carbopol® Ultrez 20 (HASNE) did not change significantly these characteristics (p N 0.05). The total flavonoids (QCT, LUT, and 3MQ) content was close to 1 mg/mL in ASNE and HASNE. Such characteristics remained similar (p N 0.05) after 90 days of storage at 4 °C. Moreover, the visual analysis did not indicate the presence of instability, such as creaming, coalescence or phase separation. TEM photomicrographs of ASNE and HASNE are presented in Fig. 1. Image 1A of ASNE showed typical appearance of oil nanodroplets with spheroid shape and well-defined edge with droplet size in a 200– 300 nm range. Fig. 1B shows individual oil droplets of nanoemulsions after dispersion into hydrogel, exhibiting a droplet size similar to the ones detected before gel thickening; however, oil droplet boundaries seem to be more irregular. 3.2. Rheological Characterization of HASNE Fig. 2 exhibits the rheological analysis of HASNE. As can be seen, HASNE behaves as a non-Newtonian pseudoplastic fluid, since the viscosity decreases with increasing shear rate. The data obtained for shear rates and shear stress were adjusted to different mathematical models to predict flow behavior. The determination coefficient (r2) determined for Bingham (0.9991), Ostwald (0.9996), Casson (0.9995), and Herschel-Bulkley (0.9611) models indicates that Ostwald model best fits flow characteristics of HASNE formulation. The flow index value (n), obtained by Ostwald equation (Ƭ = Kɣn, where n is the flow index), was 0.66, indicating a non-Newtonian fluid with pseudoplastic behavior. 3.3. Skin Flavonoids Permeation/Retention Studies Fig. 3A shows the amount of total flavonoids from ASNE retained into the porcine ear skin as function of the amount of formulation applied (in the donor compartment) after 8 h of kinetics. As can be seen, the addition of increasing amount of ASNE on the skin (10–1000 μL) led to a progressive retention of flavonoids up to approximately 2 μg/ cm2 of skin (for 100 μL of ASNE applied). After that, a plateau was reached and the amount of flavonoids retained remained almost unchanged. Fig. 3B depicts the amount of each flavonoid retained (QCT, LUT, and 3MQ) into the porcine ear skin. A similar pattern was i.e. an increasing of retention of each flavonoid up to of 100 μL of ASNE applied. Table 1 Physicochemical properties of nanoemulsions and flavonoid content (QCT, LUT, and 3MQ) just after preparation and after 90 days of storage at 4 °C. t90

t0

Droplet size (nm) Polydispersity index Zeta potential (mV) QCT (μg/mL) LUT (μg/mL) 3MQ (μg/mL)

ASNE

HASNE

ASNE

HASNE

245.9 ± 11.0 0.19 ± 0.09 −50.7 ± 3.0 319.0 ± 16.1 154.4 ± 5.5 621.2 ± 17.4

253.8 ± 3.73 0.17 ± 0.01 −48.0 ± 2.3 296.5 ± 12.0 153.7 ± 0.14 602.0 ± 18.4

288.2 ± 5.6 0.29 ± 0.05 −46.6 ± 1.21 291.0 ± 2.3 141.0 ± 3.5 595.0 ± 7.9

246.8 ± 3.3 0.22 ± 0.1 −42.5 ± 2.2 301.6 ± 12.0 156.8 ± 6.2 596.4 ± 6.6

Considering incorporation of 1.0% of dry residue of AS extract, that corresponds to 325.5 μg of QCT, 161.5 μg of LUT and 657.1 μg of 3MQ.

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Fig. 1. TEM images of the (a) nanoemulsion ASNE and of the (b) hydrogel HASNE.

In this plateau, the amount of 3MQ retained (1.0 μg/cm2) into the skin was significantly higher (p b 0.05) in comparison with the other flavonoids QCT and LUT that remained quite similar (0.3 μg/cm2). Table 2 shows the 3MQ retention into porcine skin layers from ASNE and HASNE after 8 h of application (100 μg). Whatever the formulation, 3MQ was found in both viable epidermis and dermis. A significant lower amount of 3MQ (p b 0.05) was found into epidermis from HASNE in comparison with ASNE. No 3MQ was detected in the receptor compartment of Franz diffusion cells.

obtained for HNE and positive control, indicating a non- enzymatic anti-oxidative protection of this formulation (Fig. 5A). A significant

3.4. Release Studies Fig. 4 shows the 3MQ release from ASNE and HASNE on cellulose ester membranes. A 3MQ release of approximately 85% and 53% was observed respectively for ASNE and HASNE after 8 h of kinetics, showing the effect of the gelling agent Carbopol® Ultrez 20 on slowing the release of this flavonoid from formulations. The 3MQ release flow was 3.61 μg/cm2/h for ASNE and 2.83 μg/cm2/h for HASNE. 3.5. Antioxidant Activity Studies The antioxidant potential of HASNE and HNE, estimated by TRAP and TAR assays, were showed in Fig. 5. In the TRAP evaluation, the AUC obtained for HASNE was significantly lower (p b 0.05) than AUC values

Fig. 2. Rheological profile of the hydrogel containing AS extract-loaded nanoemulsions (HASNE) showing ascendant and descendant curves.

Fig. 3. Skin retention profile of flavonoids after application of different amount of nanoemulsion. The results were presented as total flavonoid (Fig. 3A) and as individual flavonoid (Fig. 3B), being (■) QCT, (●) LUT (A) and (▲) 3MQ.

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Table 2 Skin retention of 3MQ from ASNE and HASNE (μg/cm2) into total skin, epidermis and dermis layers after 8 h of kinetics.

Total skin⁎ Epidermis Dermis

ASNE

HASNE

1.16 ± 0.23 0.90 ± 0.28 0.19 ± 0.06

0.67 ± 0.20a 0.51 ± 0.17a 0.16 ± 0.07

⁎ Stratum corneum + viable epidermis + dermis. a Statistical differences between ASNE and HASNE (p b 0.05).

increase of approximately 4.5-fold of TAR levels (p b 0.05) was observed for HASNE in comparison with HNE and the positive control (Fig. 5B). Trolox is an analog of vitamin E used in this study as a positive control [22]. Fig. 6 exhibits the skin levels of carbonyl groups, total thiols (t-SH) and thiobarbituric acid reactive substances (TBARS) before and after exposure UVA / UVB. A significantly higher level of carbonilated proteins was observed in the samples of skin exposed to UVA/UVB light without treatment as compared to HASNE treated skin samples (Fig. 6A) (p b 0.05). No differences were detected between treatments with HNE and HASNE. Concerning the levels of thiol groups (Fig. 6B), it was observed an attenuation in the depletion of these groups in the samples of treated skin with both HNE and HASNE in comparison with NIS and IS (p b 0.05). In samples treated with HNE, the levels of t-SH are close to 5.5 nmol and for samples treated with HASNE are significantly higher (8.5 nmol SH/mg of protein). For controls (untreated or subjected or not to UVA/UVB), a depletion of t-SH was significantly higher (p b 0.05), achieving approximately 3 nmol SH/mg of protein (group IS). Finally, the Fig. 6C shows a significant increase in the production of thiobarbituric acid reactive substances (TBARS) in the skin exposed to UVA/UVB irradiation. However, this TBARS production was significantly decreased in skin samples treated with HASNE (p b 0.05), showing a reduction of at least 100 nmol TBARS/mg of protein in relation to the control group treated with HNE and the group IS.

Fig. 5. Evaluation of the antioxidant potential of HNE and HASNE by determination of (A) TRAP-Total reactive antioxidant potential (A), and TAR –Total antioxidant reactivity (B), whereas Trolox as positive control. HNE: hydrogel containing blank nanoemulsion; HASNE: hydrogel containing Achyrocline satureioides extract-loaded nanoemulsion.

4. Discussion Topical application of flavonoids has been considered as a useful strategy to prevent deleterious effects of UV light on the skin [23]. In this way, we have recently described the simultaneous incorporation of the antioxidant free flavonoids (QCT, LUT, and 3MQ) from A. satureioides ethanol extract into topical nanoemulsions [16]. These compounds need to be released from formulations and located into the skin (epidermis and dermis layers) available to inhibit the reactive oxygen species produced due UV light exposure [24,25].

Fig. 4. Release profiles of 3MQ from ASNE (●˜) and HASNE (■).

The effect of the application of increasing amount of ASNE on the retention of flavonoids into the skin was first evaluated by using Franztype diffusion cells. A progressive increase of the skin retention of flavonoids was detected (up to 100 μL of formulation applied) achieving approximately 2 μg/cm2 of skin, followed by a plateau for which this amount remained almost unchanged. Various parameters may play a role on the permeation/retention of drugs through the skin, including those related with their diffusion through the layers of the stratum corneum and their physicochemical properties, such as the octanol/ water partition coefficient [26]. Our results showed that the amount of 3MQ retained could be related with its initial concentration in the formulation, i.e. a higher amount of 3MQ in the formulation (319.0 ± 16.1 μg/ml) led to a higher retention of this flavonoid in to the skin (1.25 μg/cm2) in comparison with QCT and LUT. In addition, 3MQ presented the highest partition coefficient (2.5) when compared to the QCT (1.5) and LUT (1.4) [27]. Thus, the 3MQ retention seems to be related with both a favorable concentration gradient and a higher partition coefficient of 3MQ. Topical administration of nanotechology-based delivery system may be facilitated by its incorporation into semisolid dosage forms. To this end, hydrogels prepared with acrylic-acid polymers have been used in the pharmaceutical field due to their prolonged residence time in the skin offering an opportunity for sustained drug release [28,29]. In this study, ASNE was embedded in a carbopol Ultrez 20 (a hydrophobically modified cross-linked acrylate copolymer) hydrogel (HASNE). This hydrogel exhibits non-Newtonian pseudoplastic behavior. Such a characteristic is highly desirable in topical formulations once they provide greater ease of administration, better spreadability and hence greater uniformity of the distribution of the formulation at the application site [30]. The

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Fig. 6. Evaluation of oxidative stress by determination of (A) carbonylated protein, (B) total thiols (protein and non-protein) and (C) thiobarbituric acid reactive species (TBARS), whereas HNE: skin treated with HNE; HASNE skin treated with HASNE; IS: irradiated skin without treatment and NIS: skin without treatment and no irradiation. Statistical differences were determined by Tukey test (p b 0.05); ‘a’ refers to the difference in relation to NIS, ‘b’ refers to the difference in relation to IS and ‘c’ refers to the difference in relation to HNE.

main physicochemical properties of nanoemulsions remained similar in the presence of the thickening agent and over time (at least for 90 days of storage at 4 °C). Hence, these semisolid formulations containing ASNE could be considered monodisperse and stable against flocculation/coalescence.

The effect of the carbopol® ultrez 20 on the skin retention of 3MQ, selected as a marker of A. satureioides extract, was further investigated. The total amount of 3MQ retained into the skin remained similar; however, the thickening of ASNE (at 0.15%) reduces the amount of 3MQ retained into the upper layers (stratum corneum + viable epidermis) of the skin. Such a result could be attributed to the lower amount of 3MQ released for HASNE after 8 h of kinetics. In fact, 3MQ was released faster and in a greater extent from nanoemulsions (ASNE) than from the derivative hydrogel (HASNE), indicating the influence of an additional barrier to drug release from hydrogel, due to the presence of polymerforming hydrogel, has been previously described in the literature [31, 32]. Finally, it must be mentioned that no flavonoids were detected in the receptor fluid after permeation/retention studies using full thickness skin. The antioxidant potential of HNE and HASNE was determined using TRAP and TAR assays. In these cases, the chemiluminescence related to the oxidation reaction induced by the AAPH radical is revealed by luminol. TRAP demonstrates the total antioxidant capacity of the formulation being associated with the type and concentration of the antioxidant. Therefore, a lower chemiluminescence represents a higher antioxidant capacity of the sample. TRAP results for HASNE showed a six-fold higher reduction in the chemiluminescence when compared to the positive control Trolox and HNE, demonstrating the high antioxidant capacity of the A. satureioides extract. In turn, TAR is a measure of the quality of the antioxidants that shows the relationship between the initial chemiluminescence of the AAPH-luminol system and the chemiluminescence of the sample containing antioxidants. In this study, the TAR results showed that HASNE is 4.5 times more powerful that the positive control. The antioxidant activity of A. satureioides has been related with the high flavonoid content in ethanolic extracts, such as QCT, LUT, and 3MQ [8,9,33,34]. It must be mentioned that the results observed for blank formulation (HNE) may be, at least in part, attributed to the presence of vitamin E in formulations even if the final concentration used can be considered low (0.5%) in comparison with previous literature [35,36,37]. Vitamin E was primary added as an antioxidant excipient to protect lipid components of the oil core (phospholipids and medium chain triglycerides). The protective capacity of the formulations against UV-induced skin damage was evaluated by analyzing different parameters of oxidative stress on treated and untreated skin after it had been irradiated with UVA/UVB light. UVB (290–320 nm) and UVA (320–400 nm) radiation have different energies and penetration abilities, causing different magnitudes of damage to the skin's structure [38]. In this study, oxidative damage to skin proteins was determined by quantifying the rate of carbonylated protein. Carbonylation is an irreversible protein modification that occurs when reactive oxygen species (ROS) attack some of the aminoacids, thus generating carbonyls groups (aldehydes and ketones). Carbonylated proteins create aggregates that are highly resistant to degradation, which then accumulate in dysfunctional masses of damaged proteins [39] and alter the skin's structure. These proteic carbonyls groups may be quantified by spectrophotometer because they react with 2.4-dinitrophenylhydrazine (DNPH) to form hydrazones, a colored compound. In our study, the level of carbonylated proteins in skin irradiated with UV light was significantly higher (~45 nmoles of carbonyl/ mg protein) when compared with non-irradiated skin (~25 nmoles of carbonyl/mg protein). Carbonylation of keratinocytes associated with UVA/UVB radiation has been described in other studies [40]. The skin was protected from protein damage caused by UV light when treated with both HNE and HASNE, with the carbonylated protein rates remaining at the basal level. In this parameter, the protection observed could be related with a light scattering property of nanosystems, being related to nanometric size, spherical shape, and system compounds [41]. Protein damage was also presumed by determining the levels of total thiol (− SH), considering that most –SH groups from skin are proteic. In proteins with thiol groups (− SH), the ROS may lead to the oxidation of thiol groups and consequently the loss of function.

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Nevertheless, unlike protein carbonylation, disulfide bond formation is reversible by endogenous or exogenous antioxidants (GSH, flavonoids). Our study demonstrated that A. satureioides extract preserved –SH levels in skin treated with HASNE being much higher than the –SH levels for irradiated skin and HNE-treated skin. More significantly, the skin treated with HASNE demonstrated an increase of –SH levels when compared to non-irradiated skin, meaning that HASNE reversed the protein damage caused by daily exposure to small doses of UV radiation. This reversal is probably due to the presence of QCT, LUT, and 3MQ in A. satureioides, as suggested in previous studies. For instance, the depletion of GSH, an endogenous antioxidant associated with UV light exposure is known to be reduced with topical treatment with QCT [12]. Moreover, a protective effect of HNE against disulfide bond formation was also observed. This fact once again illustrates the protection exerted by nanocarriers, probably by light scattering that prevents new protein damage. Finally, as an index of lipid oxidative damage we used the determination of thiobarbituric acid reactive substances levels (TBARS). A possible lipid oxidation product, especially malondialdehyde, reacts with thiobarbituric acid to produce a pink compound that can be determined spectrophotometrically. The HNE formulation had no protective effect in this parameter. Conversely, HASNE was able to partially protect the skin of the lipid peroxidation induced by UV light exposure. In this case, the TBARS level in the skin treated with HASNE was significantly lower than TBARS levels found in untreated irradiated skin or treated with HNE. Such a result may be attributed to flavonoids of A. satureioides extract. For instance, QCT, hesperetin, and naringenin have been shown to reduce lipid peroxidation mediated by UV radiation [11]. In summary, the results indicate that the amount of flavonoids retained in the skin is adequate to protect it from protein and lipid damage when exposed to UVA/UVB light.

[5]

[6]

[7]

[8]

[9]

[10]

[11]

[12]

[13]

[14]

[15]

[16]

5. Conclusion A. satureioides-loaded nanoemulsions imbedded in carbopol® Ultrez 20 are potentially useful to protect skin against UVA/UVB light. The main results showed that different parameters may play a role on the skin retention of flavonoids, including the amount of nanoemulsion applied on the skin, the amount of flavonoid content in the formulation, and the type of flavonoid. The incorporation of the nanoemulsions into the hydrogel did not change markedly the main physicochemical properties of nanoemulsions. The 3MQ release rate and extent were lowered when nanoemulsions were incorporated into hydrogels that exhibited a non-Newtonian pseudoplastic behavior. The overall results showed a protection of the porcine ear skin against the oxidative damage from UVA/ UVB radiation. Such a protection results from a possible additive effect between the antioxidant activity of the A. satureioides extract and nanostructured system itself, including the light scattering properties of the formulations. Acknowledgments This work was supported by the State Foundation for Research Support - FAPERGS (n° 2319-2551/14-0). J.B. wishes to thank FAPERGS for her post-doctoral scholarship. H.F.T. and J.C.M are recipients of CNPq research fellowship.

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