J. Cosmet. Sci., 63, 311–320 (September/October 2012) 311 Skin-whitening effects of Mediterranean herbal extracts by in vitro and in vivo models LUISA RIZZA, CLAUDIA BONINA, GIUSEPPINA FRASCA, and CARMELO PUGLIA, Department of Pharmaceutical Science, School of Pharmacy, University of Catania, Viale A Doria 6, 95125 Catania, Italy. Accepted for publication February 13, 2012. Synopsis Several plant extracts are able to protect skin against ultraviolet-light-induced damage and hyperpigmenta- tion in a safe way. The anti-melanogenic effect of herbal extracts seems to be related to their antioxidant activ- ity and their polyphenolic content. In this study, the skin-whitening effect of some Mediterranean species, already known for their strong antioxidant and radical scavenger activity, has been evaluated by in vitro and in vivo models. The results obtained showed that herbal extracts possessed an inhibitory effect on tyrosinase enzyme. Each extract showed a similar inhibiting activity even though it was less intensive than kojic acid and hydroquinone. Otherwise, a signifi cant higher activity than kojic acid and hydroquinone was observed when the herbal extracts were combined. Furthermore, the anti-melanogenic activity and an evaluation of skin tolerance were affected by in vivo methods. INTRODUCTION Melanin is a physiological cutaneous “sunscreen” because it plays an essential role in the protection of skin from sunlight-induced damage. However, excessive production and accumulation of melanin can cause hyperpigmentation (melasma, freckles, age spot), and it can be associated with most serious skin disease such as melanoma (1–3). Melanin production can be inhibited by the reduction of ultraviolet (UV) exposure, by physical removal of corneal layer or inhibition of melanocyte biosynthesis and metabo- lism (4). Currently, topical application of melanin synthesis inhibitors is the less inva- sive procedure to avoid skin hyperpigmentation. Until now, traditional synthetic inhibitors of tyrosinase, such as hydroquinone and kojic acid, are considered the main active ingredients in cosmetic skin-whitening products (4–6). However, topical ap- plication of cosmetic and cosmeceutical products containing these active substances can raise several safety concerns especially for long-term treatments. Actually, some of their adverse effects, such as erythema, skin irritation, dermatitis, exogenous ochrono- sis, impaired wound healing, sclera and nail pigmentation, restricted their usage in the cosmetic industry (5,7,8).

JOURNAL OF COSMETIC SCIENCE 312 In the search for novel depigmenting agents, the use of natural herbal extracts as new skin-care active ingredients has been recently emphasized. As reported by scientifi c works (5–10), several plant extracts are able to protect skin against UV-induced dam- age and to inhibit melanogenesis in a safe way, without cytotoxicity or mutagenicity effects. Nowadays, polyphenols are considered the main group of natural compounds responsible of these effects (11). The mechanism of action of polyphenols in human skin is not yet clearly understood, but it is supposed that their action is strictly corre- lated to reduction of cutaneous oxidative stress (11). In fact, it is well known that in melanocytes melanin synthesis occurs in a cascade of enzymatic reactions that converts tyrosine to melanin pigment. The key enzyme and rate-limiting reaction of this cas- cade is the tyrosinase that catalyzes three steps of melanin biosynthesis: hydroxylation of tyrosine to 3,4-dihydroxyphenylalanine (DOPA), oxidation of DOPA to dopaqui- none, and oxidation of 5,6-dihydroxyindole to indolequinone (5). Reactive intermedi- ates produced are further oxidized to form melanin by a free radical-coupling pathway (4,9). Radical species, especially reactive oxygen ones, are considered to play an impor- tant role in regulating the melanogenesis and the proliferation of melanocytes (10). Antioxidants and inhibitors of radical production may affect melanogenesis events. Consequently, as reported by Panich et al. (3), the anti-tyrosinase effects of some plant extracts appeared to be correlated with their antioxidant potential. Finally, it was sug- gested that successful skin-whitening treatments could require the use of different anti-melanogenic ingredients to achieve a more intensive effect (12). Herbal extracts often induced more intensive effects, such as antioxidant and anti-melanogenic one, when used in combination (12–15). In this study, the anti-melanogenic effects of herbal extracts obtained from typical Medi- terranean species such as Capparis spinosa, Citrus sinensis, Oryza sativa, and Olea europaea have been studied. These plants have been chosen since characterized by an interesting antioxidant activity attributed to the high level of polyphenols (16–19). The anti- melanogenic effect of each extract and their combination was evaluated by in vitro models while in vivo experiments were performed to determine their skin-whitening action and skin tolerance (skin sensitivity to UVB irradiation). MATERIALS AND METHODS TEST MATERIALS Hydroquinone, kojic acid, and other cosmetic ingredients to produce topical formulation were supplied by A.C.E.F. s.p.a. (Fiorenzuola, Piacenza, Italy). Caper buds (Capparis spinosa), blood orange (Citrus sinensis), rice grains (Oryza sativa), and olive leaf (Olea europaea) solid extracts and their mixture were supplied by Bionap srl (Skin Moon Catania, Italy). Briefl y, plants (caper buds, rice grains, olive leaf) were chopped into small pieces and then extracted with ethanol/water solution (50:50) acidifi ed to pH = 3 for citric acid for 12 h. After- ward, extracts and squeezed orange juice were fi ltered using 0.2 μm paper fi lter to remove any impurities and then were applied to a polystyrene/divinylbenzene XAD-16 column (Rohm and Haas, Philadelphia, PA). The resins were eluted with an ethanol/water solu- tion (50:50), then ethanol was removed by evaporation and the aqueous residue was spray-dried. Herbal solid extracts showed to possess similar total phenol contents (% w/w)