INHIBITION OF MELANIN CONTENT BY PUNICALAGINS 451 Figure 6. Analysis of ellagic acid levels in Melan-a cell cultures treated with ellagic acid. The HPLC was calibrated for ellagic acid with a detection limit set at 11 μg/ml (A). Melan-a cells were treated with 50 μg/ml ellagic acid for 48 h and the culture medium (B) and cell lysates (C) were analyzed for ellagic acid content by HPLC.

JOURNAL OF COSMETIC SCIENCE 452 acid, our data indicate that punicalagins act directly on melanocytes to inhibit melanin content. These results are further supported by reports from our laboratory that punica- lagins, unlike ellagic acid, do not inhibit tyrosinase activity in Melan-a cell melanocytes (17). In addition, we have also found that a combination of pomegranate extract standard- ized to 20% punicalagins and Siberian larch standardized to 80% taxifolin synergistically reduced melanin content more than pomegranate extract or Siberian larch alone (25,26). Most inhibitors of melanogenesis (e.g., arbutin, kojic acid, licorice extract, and ascorbic acid) are potent antioxidants and show anti-infl ammatory activity (22). Consistent with the antioxidant and anti-infl ammatory activity of most skin lightening ingredients, pu- nicalagins have been reported to inhibit oxidative stress and activity of the proinfl amma- tory transcription factor NFκb (23). Furthermore, because of its solubility and stability in aqueous medium and its high bioavailability characteristics, punicalagins are well suited for cosmetic formulation (24). In recent years, many publications have reported in vitro and in vivo skin lightening effi cacy of ellagic acid rich pomegranate extracts. In a double-blind placebo-controlled study with for- mulations containing synthetic ellagic acid and another with pomegranate extract containing natural ellagic acid, the authors found that topical application of synthetic or natural ellagic acid were comparable in skin lightening effi cacy (13). In another study, topical application of pomegranate juice extract mitigated melanin levels and erythema index in female subjects (14). Furthermore, it has been shown that oral intake of pomegranate extract rich in ellagic acid ameliorates skin pigmentation induced by UV irradiation (15). On the basis of these re- ports of in vivo and in vitro skin lightening effi cacy of pomegranate extracts containing ellagic acid, our fi ndings suggest that the melanin-inhibiting function of punicalagins and pome- granate extract (standardized to 20% punicalagins) could provide skin lightening benefi ts. CONCLUSIONS These results show that pomegranate fruit extract with 20% punicalagins directly reduce melanin content in Melan-a cells and is noncytotoxic at the effective concentrations. Our data combined with the recent reports on the in vivo effects of pomegranate extract suggest that pomegranate punicalagins are an attractive candidate for topical skin lightening application. ACKNOWLEDGMENTS We thank Mark Proefke, Manager, Analytical Sciences, and Barry Traband, Manager, Con- centrate Development, for their support and encouragement to undertake this project. We also like to express our sincere gratitude to Audra Davies, VP-Nutritional Product Devel- opment, and Analytical Sciences, for her encouragement and approval to conduct this re- search. We also extend our thanks to Ms. Barbara Olson for critical reading and evaluation of this manuscript. REFERENCES (1) A. Slominski, D. J. Tobin, S. Shibahara, and J. Wortsman, Melanin pigmentation in mammalian skin and its hormonal regulation, Physiol. Rev., 84, 1155–1228 (2004). (2) E. Raynaud, C. Cellier, and J. L. Perret, Depigmentation for cosmetic purposes: Prevalence and side- effects in a female population in Senegal, Ann. Dermatol. Venereol., 128, 720–724 (2001).