JOURNAL OF COSMETIC SCIENCE 64 the 3′ and 5′ positions of the B-ring. Acylation of the sugar moiety with various aromatic and aliphatic acids may also occur (2). The biological activity of ACNs is largely depen- dent on these variations in their chemical structures. The intense and attractive colors produced by ACNs have prompted interest in their uses as colorants in the food industry (3). Their potential to act as powerful antioxidants and for use in disease prevention has also been gaining increased attention (4). They have long been considered as strong antioxidants, with the ability to scavenge free radicals and ter- minate chain reactions demonstrated in many in vitro assays (5,6). Their protective effects against oxidative stress–induced damage and regulation of redox signaling pathways have also been demonstrated (7–9). Unabsorbed ACNs have also been shown to potentially act as chemopreventive agents topically in the gastrointestinal tract by preventing oxidative damage to the mucosal lin- ing (10). This topical activity may also translate to similar benefi ts when applied to the skin in an appropriate vesicle, such that they are able to react with damaging reactive oxygen species (11). Recent investigations into ACNs’ potential to prevent oxidative damage to the skin by ultraviolet (UV)-induced erythema, skin cancer, and photoaging have also demonstrated a protective effect in vitro and in vivo (12–15). Photoaging is de- fi ned as premature aging of the skin caused by repeated exposure to UV radiation, with signs including general skin deterioration and dark spots or abnormal skin pigmentation (16). Anthocyanins have shown a potential to trigger a regenerative effect on the skin (17). Moreover, they have been shown to improve psoriatic lesions in vitro and alleviate atopic dermatitis in vivo (18,19). Few studies have investigated the protective effects of ACNs when incorporated into matrices for topical delivery. However, two recent studies positively demonstrated the biological activity of ACNs when concentrated onto protein-rich matrices (20) and also when incorporated into ultradeformable liposomes (11). The results of these studies were promising however, the technology used does not easily translate into practical or commonly used delivery systems for bioactive ingredients, such as incorporation into pre-existing cosmetic formulas. In cosmetic formulations, the chemical and potential bioactive properties of ACNs could translate into antiphotoaging properties. When incorporated into some cosmetic formulations, the color imparted by these pig- ments and their stability must also be considered. We recently reported ACNs to be suc- cessful colorants of lipstick formulations with stability comparable with synthetic pigments, determined to exceed 2 years based on the accelerated testing conditions (21). Therefore, the aim of this study was to investigate the potential biological activity of ACNs when incorporated into lipstick formulations as a source of color and as an active ingredient, with ACNs selected based on our previous work. MATERIALS AND METHODS MATERIALS Elderberry, purple carrot, purple sweet potato, and red radish dried extracts were pro- vided by DD Williamson & Co., Inc. (Louisville, KY), and the purple corn and red grape skin dried extracts were provided by Artemis International (Fort Wayne, IN). The base of the lipstick formulations was purchased from MakingCosmetics, Inc. (Snoqualmie, WA)

PROPERTIES OF ANTHOCYANIN-PIGMENTED LIPSTICK FORMULATIONS 65 ingredients for the base were as follows: triglyceride, coconut oil, octyldodecanol, ozoke- rite wax, polyisobutene, castor oil, isopropyl palmitate, microcrystalline wax, lanolin oil, microcrystalline wax, synthetic wax, glycerin, DL-alpha tocopherol, and butylated hy- droxytoluene (BHT). Black lip balm containers were purchased from a local company, Bulk Apothecary (Streetsboro, OH). Compounds used were gallic acid, 2,2-diphenyl- 1-picrylhydrazyl (DPPH), BHT, mushroom tyrosinase, L-3,4-dihydroxyphenylalanine (L-DOPA), and kojic acid, purchased from Sigma-Aldrich (St. Louis, MO). Reagents used were ethanol and methanol, and were purchased from Fisher Scientifi c, Inc. (Fair Lawn, NJ). LIPSTICK SAMPLE FORMULATIONS Formulations were prepared according to our previous formulation (21) and based on recommendations in the Society of Cosmetic Chemists Monograph Number 8: Lipstick Technology (22). All dried extracts were incorporated as 8% of the fi nal weight (w/w) of each lipstick formulation based on preliminary data (21). Dried extracts were weighed and ground in castor oil at a 1:3 ratio (pigment:oil) by mortar and pestle. Silica was in- cluded at 1% of the fi nal weight (w/w) to increase uniformity in the fi nal products. The lipstick base was heated in a water bath at 70°C, and the preground pigment extracts were then added to the hot lipstick base and homogenized. The lipstick formulas were then poured directly into lip balm containers and cooled at 4°C until completely solid. CHARACTERIZATION OF ACNS BY HIGH-PRESSURE LIQUID CHROMATOGRAPHY The ACNs of the extracts used in these lipstick formulations were evaluated by reverse- phase high-performance liquid chromatography. The system (Shimadzu Corporation, Co- lumbia, MD) was composed of an LC-20AD prominence liquid chromatograph and an SPD-M20A prominence diode array detector coupled to an LCMS-2010 mass spectrom- eter (Shimadzu Corporation). LCMS solution Ver 3.30 software was used to collect and evaluate data. Anthocyanin separation was achieved on a reversed-phase 3.5-μm Symme- try C18 column (4.6 × 150 mm Waters Corp., MA) fi tted with a 4.6 × 150-mm Sym- metry 5 microguard column (Waters Corp.) with a binary gradient of 4.5% (v/v) formic acid in water (solvent A) and 100% acetonitrile (solvent B) at a fl ow rate of 0.8 mL/min. Solvent gradient followed 10–30% B from 0 to 30 min. Spectral information was col- lected from 260 to 700 nm, and elution was monitored at 280 and 520 nm. For MS analysis, 0.2 mL/min fl ow was diverted to the MS and ionized under positive ion condi- tions using an electrospray probe. Data were initially monitored using a total ion scan from m/z 200 to m/z 1,200 and then with selective ion monitoring at m/z 271 (pelargo- nidin), m/z 287 (cyanidin), m/z 301 (peonidin), m/z 303 (delphinidin), m/z 317 (petuni- din), and m/z 331 (malvidin). SPECTROPHOTOMETRIC ANALYSIS OF TOTAL MONOMERIC ACN CONTENT The total monomeric ACN content for the extracts and lipstick formulations was measured in 1-cm cuvettes using a spectrophotometer (UV-2450 spectrophotometer Shimadzu, Kyoto, Japan) using the pH differential method as described by Giusti and