Regulatory Toxicology and Pharmacology 69 (2014) 71–77

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Photoprotective characteristics of natural antioxidant polyphenols Roberto Stevanato ⇑, Mariangela Bertelle, Sabrina Fabris Department of Molecular Sciences and Nanosystems, University Ca’ Foscari of Venice, Dorsoduro 2137, 30123 Venice, Italy

a r t i c l e

i n f o

Article history: Received 16 December 2013 Available online 5 March 2014 Keywords: Polyphenols Antioxidants Sunscreens

a b s t r a c t Fourteen natural polyphenols belonging to the classes of stilbenes, flavonoids and hydroxycinnamic acid derivatives, have been investigated in order to verify the combination of their photoprotective characteristics with their antioxidant properties. To this purpose, sun protection factor (SPF), UVA/UVB ratio and critical wavelengths (kc), have been considered to evaluate photoprotection capacity, while inhibition of lipid peroxidation has been adopted as a reliable measure of the antioxidant properties. The results obtained indicate that a large number of these natural phenol derivatives show both antioxidant activity and photoprotective characteristics and, as a consequence, they could be interesting components for pharma-photoprotection formulations. In fact, these compounds associate to a preventive function, linked to UV filtering properties, an effective action, correlated to antioxidant capacity of contrast towards UV-induced ROS injury. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction The exposure to UV radiation can generate harmful reactive oxygen species (ROS) in the skin. In particular, UVB radiation (290–320 nm) penetrating the skin to a depth of 160–180 lm, can cause erythema and sunburns and trigger off the induction of oxidative stress, DNA damage and premature aging of skin (De Gruijl and Van der Leun, 1994; Ichihashi et al., 2003; Mukhtar et al., 1996). UVA radiation (320–400 nm) can penetrate deeper into the epidermis and dermis of the skin (around 1 mm) and advance the generation of singlet oxygen and hydroxyl-free radicals, which can harm proteins, lipids and DNA (Di Giovanni, 1992; Lavker et al., 1995a,b; Lavker and Kaidbey, 1997). Furthermore, UVA is 10 times more efficient than UVB at causing lipid peroxidation (Morliere et al., 1995). Usually, endogenous enzymatic and nonenzymatic antioxidants (AOs) are able to inhibit the action of ROS; but sometimes excessive and chronic exposure to UV radiation makes these defences inadequate causing many adverse effects like premature skin aging and melanoma (Nichols and Katiyar, 2010). In order to reduce the risk of injury, more effective action is the filtering of sunny rays using sunscreen capable to absorb radiant energy in UVB and UVA range. Chemical sunscreens are usually organic aromatic compounds conjugated with carbonyl group which ⇑ Corresponding author. Fax: +39 0412348594. E-mail addresses: [email protected] (R. Stevanato), [email protected] (M. Bertelle), [email protected] (S. Fabris). http://dx.doi.org/10.1016/j.yrtph.2014.02.014 0273-2300/Ó 2014 Elsevier Inc. All rights reserved.

absorb ultraviolet rays and release lower energy rays; in this way they prevent the skin from damaging effects of UVB and UVA (Rai et al., 2012). However, UV absorption may activate organic sunscreens and they may consequently interact with cutaneous molecules, causing adverse skin reactions like dermatitis or photosensitivity reactions (Dromgoole and Maibach, 1990). Thus, in recent years the great interest of photoprotection is addressed in using natural products for their higher tolerability and for their negligible environmental impact (Nichols and Katiyar, 2010). Flavonoids, a class of secondary plant phenolics, found in fruits, vegetables, red wine and tea protect plants from solar UV radiation (Rozema et al., 1997; Burchard et al., 2000) and scavenge UV generated ROS (Shirley, 1996; Bravo, 1998; Nichols and Katiyar, 2010; Gregoris et al., 2011). In fact, flavonoids have the ability to reduce the oxidative damage caused by short solar wavelengths and reduce the risk of generation of ROS by decreasing the penetration of UVB radiation to sensitive leaf targets (Kytridis and Manetas, 2006; Kotilainen et al., 2008; Owens et al., 2008; Agati et al., 2009). Several studies have found that resveratrol (trans-3,5,4’-trihydroxystilbene), a naturally occurring polyphenolic phytoalexin found in grapes, red wine, peanuts, mulberries and fruits, is characterized by high antioxidant properties, imparts skin cancer chemopreventive action and prevents allergic diseases (Jang et al., 1997; Carbo et al., 1999; Fabris et al. 2008; Singh et al., 2011). Long-term studies have demonstrated that topical application with resveratrol (both pre- and post-treatment) results in inhibition of UVB-induced tumor

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R. Stevanato et al. / Regulatory Toxicology and Pharmacology 69 (2014) 71–77

incidence and delay in the onset of skin tumorgenesis (Baumann, 2009). However (Regev-Shoshani et al., 2003) demonstrated that resveratrol is susceptible to enzymatic oxidation, while piceid, its glycosylated form, is resistant and maintains its beneficial antioxidant capacity and biological properties. Hydroxycinnamic acid compounds, caffeic acid (CA) and its homologues and derivate, found in plants, fruits, vegetables, coffee and wine, are considered good candidates for UVB attenuators (Harborne and Williams, 2000; Tattini et al., 2004). Considering that: (i) UV filtering ability of polyphenols is correlated to the absorption spectra in the UV region; (ii) antioxidant properties, i.e. the stability of the (poly)phenoxy free radical depends on the possibility of delocalization of free electrons on the molecule, i.e. mainly on conjugated double bonds which absorb at UV high wavelengths; (iii) natural flavonoids, hydroxycinnamic acid derivatives and stilbenes are important components of the diet and, as a consequence, they and their metabolites rarely give sensitization effects, we studied the possible use of these products in photoprotective formulations, correlating the antioxidant power of these three classes of natural polyphenols, measured by the method of lipid peroxidation (Fabris et al., 2013), with their sunscreen ability, i.e. the factors of UVB and UVA protection. For this purpose, we measured: (1) the sun protection factor (SPF), universal indicator related to UVB and short UVA radiations; (2) the UVA/ UVB ratio and the critical wavelength, two parameters giving an indication of the UVA filtering properties. The results obtained indicate that a large number of these natural phenol derivatives show both good antioxidant activity and photoprotective characteristics and, as a consequence, they could be interesting components for pharma-photoprotective formulations. 2. Materials and methods 2.1. Materials All chemicals were reagent grade and were supplied from Sigma Chemical Co. (USA). Trans-piceid and trans-resveratrol, with a purity grade higher than 99%, have been supplied by the Istituto Agrario di S. Michele all’Adige (IT) (Ravagnan, Pat. EP 1 292 320). The UV absorbance spectra were determined at 10 lM in ethanol by a double-beam UV–VIS (Shimadzu UV-1800) spectrophotometer. 2.2. SPF determination Sun protection factor (SPF) is the universal indicator introduced by (Sayre et al., 1979) for describing the efficiency of sunscreen products against sunburn caused by UVB and short wavelength UVA (320–340 nm). On the assumption that the UV protection of a sunscreen depends by its absorption characteristic and concentration, in vitro methods have been introduced (Diffey and Robson, 1989; Reece et al., 1992; Springsteen et al., 1999). In vitro SPF is calculated as follows (Diffey and Robson, 1989):

P400 nm nm SðkÞ  EAðkÞ SPF ¼ P400 k¼290 nm SðkÞ  EAðkÞ  TðkÞ k¼290 nm

ð1Þ

where S is the solar spectral irradiance, EA is the erythemal action spectrum and T is the spectral transmittance. S and EA are given by literature (Diffey and Robson, 1989), while T is measured for every sunscreen. Sunscreen products are classified according to their SPF values as it follows: ‘‘minimal sun protection products’’ if SPF is from 2 to under 12; ‘‘moderate sun protection products’’ when SPF is from 12 to under 30, while ‘‘high sun protection products’’ if SPF value is P30 (FDA, 2001).

For SPF determination we used the calibrated step film model of Herzog et al. (2004), used also in Ciba Sunscreen Simulator (www.ciba.com/SUNSCREENSIMULATOR/), where the inhomogeneity of the film is introduced mathematically. In this case T is given by:

TðkÞ ¼ g  10eðkÞcdð1f Þ þ ð1  gÞ  10eðkÞcd½gf =ð1gÞþ1

ð2Þ

where d is the average thickness of the step film and it coincides with 20 lm (corresponding to an application in vivo of 2 mg/cm2); e(k) is the molar extinction coefficient, c is the molar concentration of the sunscreen; g and f are two parameters describing the structure of the step film. Herzog found that for all oil/water emulsions, g = 0.269 and f = 0.935. 2.3. UVA/UVB ratio and critical wavelength measurements Because there is not agreement upon a method to measure the protection against UVA (Boots the Chemist Ltd, 1991), we used the UVA/UVB ratio (Boots the Chemist Ltd, 1991) and the critical wavelength (Diffey, 1994), the most used methods. The first one is the ratio of the average extinctions in the UVA and UVB range and it is given by the following expression:

R 400 UVA=UVB ¼ R320 320 290

lg½1=TðkÞdðkÞ= lg½1=TðkÞdðkÞ=

R 400 320

dðkÞ

290

dðkÞ

R 320

ð3Þ

According to Boots the Chemist Ltd, a low UVA protection is obtained with a UVA/UVB ratio 0.8 (4 UVA Star Rating). The second method is the critical wavelength (kc) (Diffey, 1994) which determines, from 290 nm to kc, 90% of the integral of the absorption spectrum from 290 to 400 nm, that is:

Z

kc

290

lg½1=TðkÞdk ¼ 0:9 

Z

400

lg½1=TðkÞdk

ð4Þ

290

A five-point scale analogous to the UVA Star Rating has been introduced by Diffey (1994) to classify sunscreens as follows: kc < 325 nm (0 Broad Spectrum Rating), 325 6 kc < 335 nm (1 Broad Spectrum Rating), 335 6 kc < 350 nm (2 Broad Spectrum Rating), 350 6 kc < 370 nm (3 Broad Spectrum Rating), kc P 370 nm (4 Broad Spectrum Rating). 2.4. Inhibition of lipid peroxidation The antioxidant activity of phenol derivatives was studied in terms of inhibitory action on peroxidation of linoleic acid in SDS micelles because, in our opinion, this method, more than others, mimes the efficacy of an antioxidant compound to prevent oxidative damage on cell membrane or lipoproteins by ROS injures. The procedure is the same followed in Fabris et al. (2008); in particular, the peroxidation was initiated by the thermolabile water-soluble azo initiator 2,2’-azobis[2-(2-imidazolin-2-yl)propane] (ABIP) and measured as rate of oxygen consumption. In a typical experiment, to the solution containing SDS micelles (50 mM) and linolic acid (10 mM) in phosphate buffer (50 mM, pH 7.4), previously equilibrated with air and kept at constant temperature (37 °C) in a sealed thermostated vessel, the azo initiator ABIP (4 mM final concentration) was injected and the oxygen consumption was monitored by a Clark electrode. After few minutes, an ethanol solution of antioxidant was added and the different oxygen consumption rate was recorded. Every experiment was in triplicate.

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Fig. 1. UV spectra in the range 290–400 nm of: (a) stilbenes and catechin; (b) flavonoids; (c) hydroxycinnamic acids derivatives, at 10 lM concentration.

In the phase of lipid peroxidation propagation, initiated by ABIP, the antioxidant capacity was calculated as the ratio of the different slopes of oxygen consumption before and after the addition of the antioxidant. The result is expressed as IC50, that is the antioxidant concentration (lM) that halves the rate of oxygen consumption due to the peroxidation process. Therefore, low IC50 values indicate compounds characterized by high antioxidant properties and vice versa.

3. Results 3.1. UV measurements Fig. 1 shows UV spectra, in the range 290–400 nm, of 14 different natural polyphenols present in fruit, vegetables and propolis (Gregoris and Stevanato, 2010; Fabris et al., 2013) and of catechin, keep as reference for its acknowledged high antioxidant characteristics. In particular, UV spectra of piceid and resveratrol (Fig. 1a) are quite similar with high specific absorbance values between 290 and 350 nm and two absorption maxima at 306 nm

(eR306 = 34,320 M1cm1; ePC306 = 28,920 M1cm1) and 321 nm (eR321 = 32,920 M1cm1; ePC321 = 28,800 M1cm1), respectively. UV flavonoids spectra (Fig. 1b) show that quercetin, kaempferol and galangin are characterized by absorptions at relatively high wavelengths with respect to the other flavonoids and, in general, to all polyphenols here studied (Table 1). Their spectra show an absorbance peak between 360 and 375 nm (eQ,375 = 27,300 M1cm1; eK,365 = 27,820 M1cm1; eG,360 = 18,140 M1cm1), while for apigenin and chrysin maximum absorbance in the k range 310–330 nm (eA,330 nm = 28,820 M1cm1; eCR,310 nm = 21,020 M1cm1) was found. On the other hand, naringenin and pinocembrin show relatively low absorbance values in the k range 310–400 nm (e 6 18,000 M1cm1) and higher at k < 290 nm (eN,290 = 30,150 M1cm1; eP,290 = 39,270 M1cm1). Among hydroxycinnamic acid derivatives (Fig. 1c), caffeic acid (AC) and its derivatives (CP and DC) show relative maximum absorbance in the k range 320–330 nm (eAC,320nm = 21,340 M1cm1; eCP,330nm = 24,150 M1cm1; eDC,330nm = 21,710 M1cm1), while maximum absorbance of ferulic and coumaric acid are shifted at lower wavelengths, k < 290 and 310 nm, respectively,

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Table 1 Spectrophotometric, antioxidant and sunscreen characteristics of the investigated polyphenols (stilbenes, flavonoids and hydroxycinnamic acid homologues). Abbr.

SPF

UVA/UVB

kc (nm)

kmax (nm)

IC50 (lM)

Stilbenes

Resveratrol Piceid

R PC

19.2 9.9

0.28 0.31

340 345

305 305

1.00 ± 0.09 1.00 ± 0.09

Flavonoids

Catechin Quercetin Kaempferol Galangin Apigenin Naringenin Chrysin Pinocembrin

CT Q K G A N CR P

7.3 10.3 24.9 16.2 28.8 12.3 18.6 16.0

0.62 0.93 0.76 0.71 0.66 0.54 0.6 0.48

385 385 385 385 380 380 380 380

290 375 365 360 330 290 310 290

2.0 ± 0.2 1.6 ± 0.2 2.0 ± 0.2 3.0 ± 0.3 84 ± 8 56 ± 5 100 ± 9 110 ± 10

Hydroxycinnamic acid homologues

Coumaric acid Ferulic acid Caffeic acid Caffeic acid phenylethyl ester Dimethyl caffeic acid

ACM AF AC CP DC

9.3 11.9 28.0 15.8 16.6

0.17 0.27 0.43 0.52 0.52

335 345 365 370 370

290 310 320 330 330

25 ± 1 2.9 ± 0.3 0.50 ± 0.05 0.50 ± 0.05 0.20 ± 0.02

and are characterized by relatively lower molar absorbance values (eACM,290 nm = 18,800 M1cm1; eAF,310 nm = 16,500 M1cm1). 3.2. Polyphenols as photoprotectors The high molar extinction coefficients of all these natural polyphenols (10,000 6 e315 nm 6 32,000 M1cm1) make these molecules good UV absorbers and, as a consequence, they could be useful active component in sunscreen formulations, singularly or in combination between them. In order to verify this possibility, we determined their sun protection factor (SPF), UVA/UVB ratio and critical wavelength (kc), for a product concentration of 7% (w/v), a reference value already used for these evaluations (Gregoris et al., 2011). The results of these parameters for all polyphenols are reported in Table 1. 3.2.1. SPF factor In Fig. 2, SPF values for all the considered polyphenols are reported. The values of two references, catechin (CT), known antioxidant molecule but with minimal photoprotective characteristics,

and tinosorb S (TS), known sunscreen drug used in skin care market, are also shown. As regard the stilbenes, resveratrol can be taken into consideration as moderate UVB photoprotector with a SPF value slightly higher than that of tinosorb S (SPF  20), while piceid shows a minimal SPF protection. This difference is due to the presence of the glycoside group which entails a higher molecular weight; however, this group makes this stilbene more hydrophilic and therefore more soluble in the aqueous cellular milieu, protecting highly reactive soluble functional groups (OH-group) (Pearse et al., 2005; Fabris et al., 2008). As a conclusion, a photoprotective formulation combining the two stilbenes could be of interest, for their different lipophylicity that permits their to act at different levels in cells and tissues, and for their extraordinary antiinflammatory and antioxidant characteristics (Fabris et al., 2008). Among flavonoids, apigenin and kaempferol show the highest UVB protection values (SPF = 28.8 and 24.9, respectively), followed by chrysin, galangin, pinocembrin and naringenin with values comparable to that of tinosorb S. These results highlight how all the studied flavonoids, with the exception of quercetin, which

Fig. 2. SPF values of studied polyphenols, catechin and tinosorb S, at 7% (w/v) concentration. Dotted lines at 2 and 12 SPF values delimit the area of ‘‘minimal’’ (from 2 to under 12) and ‘‘moderate’’ (from 12 to under 30) sun protection properties (see text).

R. Stevanato et al. / Regulatory Toxicology and Pharmacology 69 (2014) 71–77

manifests a lower UVB protection (SPF value 10), may be taken into consideration as UVB filters. Regarding hydroxycinnamic acid derivatives, caffeic acid shows the highest SPF value (28.0), comparable with that of apigenin, while its dimethyl (DC) and phenylethyl ester (CP) derivatives, for their higher molecular mass due to methyl and phenylethyl groups, have values similar to that of tinosorb S: all these three compounds give a moderate UVB protection, unlike coumaric and ferulic acid, for which minimal properties were found. 3.2.2. UVA/UVB ratio and critical wavelength The UVA photoprotection properties of the studied compounds are summarized in Fig. 3, where the UVA/UVB ratio and critical wavelength values (kc) are estimated in terms of UVA star rating and kc broad spectrum rating, respectively (see Section 2). It can be seen that in every case, with the exception of quercetin, the kc broad spectrum rating is higher by one or sometimes two categories than UVA Star Rating. In particular, in the case of kc broad spectrum rating, stilbenes are characterized by a rating value of 2, such as cumaric and ferulic acids; caffeic acid shows a rating of 3, while all others investigated polyphenols evidence a rating value of 4, so indicating that they could be considered very good UVA photoprotective compounds. On the other hand, by UVA/UVB ratio parameter different results were found. In fact both stilbenes show moderate UVA screen properties (1 UVA star rating) so they cannot be regarded as efficient UVA filters. Between flavonoids, quercetin shows the higher UVA photoprotection (4 stars), followed by kaempferol, galangin and apigenin (3 stars like tinosorb S) and chrysin, naringenin, pinocembrin (2 stars). Among hydroxycinnamic acid derivatives, caffeic acid (AC) and its dimethyl (DC) and phenylethyl ester (CP) derivatives give a good UVA protection (2 stars), while ferulic (AF) and coumaric (ACM) acid show moderate (1 star) and low (0 star) protection, respectively. 3.3. Inhibition of lipid peroxidation Inhibition of lipid peroxidation represents a good measure of the antioxidant properties of a molecule, better than other used methods (Folin–Ciocalteu, DPPH, enzymatic assay, etc.), because

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it reproduces in vitro the action that antioxidants carry out in vivo (Fabris et al., 2008). It has been demonstrate (Gregoris et al., 2010) that, in the case of polyphenols, flavonoids in particular, a good correlation takes place between resonance limit formula of the phenoxy radical, first step of the antioxidant activity of a polyphenol, and its absorbance spectrum. In this contest, we compare their sun protective capacities and their antioxidant properties, expressed as IC50, in order to individuate natural polyphenols, which can simultaneously act as UV filters and antiradicals in skin protection. From Fig. 4, where IC50 values of all the polyphenols here investigated are reported, it appears that both stilbenes can be considerated good antioxidant molecules having a IC50 value two times lower than that of the catechin taken as reference. As regard to flavonoids, quercetin and, in lower amount, kaempferol and galangin, show higher antioxidant activity, on the contrary of apigenin, naringenin, chrysin and pinocembrin, for which low antioxidant properties have been found. Caffeic acid (AC) and its derivatives (CP and DC) exhibit very high antioxidant activity, greater than that of catechin, while a value similar to that of reference for ferulic acid has been found; on the contrary, coumaric acid is characterized by low antioxidant activity. 4. Discussion Experimental data show that, as expected, all three classes of phenol derivatives – stilbenes, flavonoids and hydroxycinnamic acid derivatives – show high absorptivity in the UV range, with different values of molar absorbance and maximum wavelength, depending on their molecular structure and mass. This is due to the presence of multiple conjugate double bonds that permits a wider delocalization of the unpaired electron produced by the antioxidant action. The three parameters used to classify the sunscreen effectiveness of a molecule, even if all ones based on the UV molecular absorption spectra, originate from different rationales (see Section 2) and, as a consequence, give different results depending on the different spectra shapes that correlate to the molecular structure. We thought that taking into account three different methods, a better and more complete information about the sunscreen characteristics of every compound can be obtained. By all the data of sunscreen characteristics here discussed (SPF, UVA/UVB ratio and critical wavelength kc), it can be suggest that a

Fig. 3. UVA star rating (dotted rods) and kc broad spectrum rating (empty rods) for all investigated compounds, at 7% (w/v) concentration.

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Fig. 4. Antioxidant activity of polyphenols expressed as 50% inhibitory concentration (IC50) of lipid peroxidation. IC50 values under 3.5 lM, represented by the dotted line, indicate compounds characterized by high antioxidant characteristics.

co-formulation of quercetin with apigenin or kaempferol or caffeic acid could assure a high sun protection over all UV wavelengths by virtue of their high and complementary absorbance peaks; these formulations could be more effective as broad spectrum protection than tinosorb S. In the light of the antioxidant properties, a co-formulation of quercetin and caffeic, or its derivative CP and DC, is more advisable, since in addition to providing a high protection factor over the entire UV spectrum, these molecules can also deliver high antioxidant activity useful against oxygen species and UV-induced oxidative DNA damage. The use of natural products, extracted from vegetables and fruit, but also from their production castaways, instead of those industrially produced, is better for environmental and toxicological reasons. Furthermore, natural polyphenols, being present in the diet, give less sensitization effects than chemically-produced sunscreen, such as their metabolites and, probably, their photoinduced products (Nichols and Katiyar, 2010). Moreover, it has been demonstrate that topical formulations of polyphenols or polyphenolsenriched extracts appear to be very efficacious against oxidative stress and DNA damaging effect of UV radiations on the skin. In particular, it has found that resveratrol inhibits UVB-induced skin tumor initiation, promotion and progression without induced toxicity (Jang et al., 1997; Aziz et al., 2003); plant polyphenols can reduce inflammation, oxidative stress, DNA damaging effects of UV radiation in the skin; furthermore plant polyphenols associate phototoxicity against skin tumor cells and photoprotection of non-tumor cells (Korkina et al., 2009; Nichols and Kativar, 2010); green tea extracts applied topically prior to psoralen plus UVA treatment result in an almost complete abrogation of the induced erythema (Zhao et al., 1999). As a conclusion, the above reported co-formulations can be excellent components for photoprotector products. In fact, they can slow down UV-induced ROS injuries associating to a preventive function, correlated to UVA and UVB filtering properties, an effective action, due to their good antioxidant activity. Conflict of interest None.

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