J. Cosmet. Sci., 64, 445–453 (November/December 2013) 445 Inhibition of melanin content by Punicalagins in the super fruit pomegranate (Punica granatum) JATINDER RANA, GANESH DIWAKAR, LISA SAITO, JEFFREY D. SCHOLTEN, and TIMOTHY MULDER, Analytical Sciences, Research and Development, Amway Corporation, Ada, MI 49355 (J.R., G.D., J.D.S., T.M.), and Nutrilite Division of Amway, Concentrate Development, Lakeview, CA 92567 (L.S.). Accepted for publication May 8, 2013. Synopsis Current efforts to develop effective skin lightening products through the inhibition of melanin production have focused on compounds that inhibit the function and activity of tyrosinase, the rate-limiting enzyme in the melanin biosynthesis pathway. Synthetic tyrosinase inhibitors, such as hydroquinone, kojic acid, and arbutin, have been reported to cause skin irritation or acute dermatitis, raising concerns about the safety of these compounds. As a result, there is a need for safe natural ingredients that show effective skin lightening. In this report, we have identifi ed a natural ingredient, pomegranate fruit extract, that inhibits melanin production in melanocytes and shows potential for use as a cosmetic skin lightening agent. In addition, we have identifi ed a polyphenolic compound, punicalagins, as the active melanin inhibitor in pomegranate fruit extract based on its capacity to directly inhibit melanin production. INTRODUCTION Skin pigmentation is primarily determined by the amount of melanin produced by melano- cytes in the skin epidermal–dermal junction. Melanin biosynthesis is catalyzed by tyrosinase, the rate-limiting enzyme in the melanin biosynthetic pathway in melanocytes. Tyrosi- nase catalyzes two steps in melanin synthesis, the oxidation of L -tyrosine to L -dopa (L-3, 4-dihydroxyphenylalanine), and L -dopa to dopaquinone and its subsequent conversion to dopachrome, which autocatalyzes to a series of intermediate products to form the brown-black pigment, melanin (1). Skin pigmentation due to synthesis and dispersion of melanin in the epidermis is of great cosmetic signifi cance. Lower amounts of melanin in the skin epider- mis signify lighter skin, whereas higher amounts of melanin are found in darker skin. Current efforts to develop effective skin lightening products have focused on agents that inhibit the function and activity of tyrosinase. Synthetic tyrosinase inhibitors, such as hydroquinone, kojic acid, and arbutin, have been shown to cause skin irritation or acute dermatitis raising concerns about the safety of these compounds (2,3). As a result, there Address all correspondence to Jatinder Rana at jatinder.rana@amway.com.

JOURNAL OF COSMETIC SCIENCE 446 is a need for safe natural ingredients that show effective skin lightening. Recently, there have been extensive reports in the literature that plant-derived polyphenols and tannins inhibit the catalytic activity of tyrosinase, thus providing a natural alternative to syn- thetic tyrosinase inhibitors (4). Pomegranate (Punica granatum) extracts have been used in ancient folk medicines by nu- merous cultures (5,6). Extracts from different portions of the plant such as seeds, peels, and leaves have been reported to exhibit strong antimicrobial (7) and antioxidant activity (8). It has also been shown that ingestion of pomegranate juice signifi cantly reduces the progression of atherosclerosis in hypercholesterolemic mice (9). Several of the health- promoting properties of pomegranate fruit have been attributed to its polyphenolic com- pounds and tannins. Prominent among them are the hydrolyzable ellagitannins that, upon hydrolysis, produce ellagic acid (10) a compound known to possess anti-infl ammatory, antitumorigenic, antiproliferative, and antiapoptotic activities (11). In recent years, ellagic acid has been shown to inhibit tyrosinase activity and melanin production (12). Topical application of ellagic acid to UV-induced hyperpigmented skin of guinea pigs and human subjects signifi cantly reduced skin pigmentation (12–14) and oral dosing of ellagic acid to guinea pigs signifi cantly lowered UV-induced skin hyper- pigmentation (15). In addition, a study showing antioxidant, antiglycation, and tyrosi- nase inhibiting activities of a polysaccharide fraction from pomegranate has been reported (16). On the basis of this evidence, we examined the effects of pomegranate fruit extract and its predominant polyphenol, punicalagins (Fig. 1), on the inhibition of melanin pro- duction in melanocytes. MATERIALS AND METHODS REAGENTS Gallic acid [97.5–102.5% (titration) Sigma-Aldrich, St. Louis, MO] and punicalagins (PhytoLab GmbH & Co. KG, Germany) were used as standards for phytochemical analysis. Figure 1. Chemical structure of punicalagins.