SKIN-WHITENING EFFECTS OF MEDITERRANEAN HERBAL EXTRACTS 315 STATISTICAL ANALYSIS All data obtained were submitted to statistical analysis. Evaluation of the statistical sig- nifi cances was performed by Student’s t-test for in vitro data. All statistical comparisons of in vivo data were evaluated using repeat measure analysis of variance followed by the Bonferroni–Dunn post hoc pair-wise comparison procedure (21). A p value of less than 0.05 was considered signifi cantly different. RESULTS The anti-tyrosinase activity of kojic acid, hydroquinone, herbal extracts from caper buds, blood orange, rice grains, and olive leaf, and their combination was evaluated by in vitro dopachrome method. As reported in Table II, all the active ingredients possessed an in- hibitory 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 ex- tracts were combined. The results of in vivo studies corroborated the in vitro fi ndings. In fact, the topical application of the formulations containing hydroquinone, kojic acid, and the mixture of herbal ingredients induced a signifi cant reduction of the skin melanin index. The trends in mean MI variation (ΔMI) vs. time for subjects are reported in Figure 1. After the fi rst week of sunlamp treatment, a signifi cant increase of skin pigmentation was observed in the control skin sites (not treated), whereas treated sites showed lower MI values, as evidenced by AUC values reported in Figure 2. Finally, from the inhibition of skin pigmentation (PI) values showed in Figure 3, it was observed that hydroquinone (PI = 69%) and the mixture of herbal extracts (PI = 75.9%) were more effective to inhibit skin pigmentation than kojic acid (PI = 52.3%). The photosensitizing effect of each skin-whitening agent was also evaluated by in vivo study. As reported in Figure 4, hydroquinone and kojic acid (photosensitivity of 48% and 32% respectively) induced comparable skin sensitivity to UV light after topical applica- tion (4 weeks) but more intensive than mixture of herbal agents (photosensitivity of 17%). Table II Inhibitory Activity (IA) Percentage of Tyrosinase Obtained from Kojic acid, Hydroquinone, Caper Buds, Blood Orange, Rice Grains, and Olive Leaf Extracts and Their Mixture at the Concentration of 50 μg/ml. Sample IA % Kojic acid 43.7 ± 3.8 Hydroquinone 30.2 ± 3.1 Caper buds (Capparis spinosa) extract 18.2 ± 2.7a Blood orange (Citrus sinensis) extract 22.0 ± 2.9a Rice grains (Oryza sativa) extract 17.9 ± 2.3a Olive leaf (Olea europaea) extract 23.1 ± 2.5a Mixture of herbal extracts 60.2 ± 4.1a a Signifi cantly different compared to kojic acid and hydroquinone ( p 0.05).

JOURNAL OF COSMETIC SCIENCE 316 DISCUSSION The in vitro data obtained in this study showed that the active compounds contained in typical Mediterranean plants (Capparis spinosa, Citrus sinensis, Olea europaea, and Oryza sativa) possessed anti-tyrosinase activity and they could be considered natural skin-whitening ingredients. Moreover, their effects were increased and potentiated when herbal extracts of each plant were mixed. The in vivo models used in this work validated the skin- whitening effect and the skin tolerance of this combination in comparison with kojic acid and hydroquinone which are well-known active ingredients used in cosmetic fi eld. Figure 1. Trend of skin melanin index (ΔMI) vs. time (days) over the monitoring period of 4 weeks for skin sites treated with formulation A (hydroquinone), formulation B (kojic acid), and formulation C (mixture of herbal extracts) or no treated (Control). Figure 2. Mean area under curve values (AUC ± DS) obtained from skin sites treated with formulation A (hydroquinone), formulation B (kojic acid), and formulation C (mixture of herbal extracts) or no treated (control) over the monitoring period of 4 weeks *p 0.05 (no signifi cantly different) vs. formulation A.