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
JOURNAL OF COSMETIC SCIENCE 66 Wrolstad (23). Anthocyanins were extracted from the lipsticks using equal parts: acetone (70%), acidifi ed ethanol (0.01% HCl), and acidifi ed deionized distilled water, followed by phase partition with chloroform as previously described (21). Sample extracts were diluted in pH 1.0 buffer (KCl-HCl) and in pH 4.5 buffer (sodium acetate) and equili- brated for 15 min. The ACN content, expressed as cyanidin-3-glucoside, was determined using the follow- ing equation: q q1,000  ž ­ ž ® pH1.0 pH4.5 Abs Abs DF mg¬ Total monomeric anthocyanin content L ƥqd ( 1) where DF = dilution factor, ε = molar absorptivity of 26,900, and d = path length (1 cm), and the molecular weight of the monomeric ACN content is 449.2. TOTAL PHENOLIC CONTENT The Folin–Ciocalteu method was used to estimate the total phenolic content of the for- mulas containing ACNs, based on the methods described in the literature (24). Lipstick formulas were dissolved in methanol and briefl y sonicated. After sample preparation, they were equilibrated in the dark at room temperature for 2 h before measuring absorbance readings at 765 nm with a spectrophotometer (UV-2450 spectrophotometer Shimadzu). Analyses were conducted in triplicate. Gallic acid was used as a positive control to deter- mine a standard curve for the test. Linear regression was used to determine a standard curve for the absorbance at 765 nm of the gallic acid solutions (R2 = 0.99). The results were reported as mg polyphenolic/L of extract solution as gallic acid equivalents (GAEs). IN VITRO UV ABSORPTION AND SUN PROTECTION FACTOR (SPF) CALCULATION In vitro SPFs for the lipstick formulas were determined based on the methods described by Sayre et al. (25) and Dutra et al. (26) with modifi cations based on the Cosmetics Europe-The Personal Care Association revised method (27). Aliquots of each solution were then pipetted into a UV microwell plate, in eight replications. Absorbance readings were measured by using a SpectraMax190 plate reader (Molecular Devices, Sunnyvale, CA) across the 290- to 400-nm UV wavelength range, at 1-nm increments and blanked against ethanol. The absorbance values were then averaged, and the standard deviations (SDs) were calculated for each sample. The in vitro SPF values were determined according to the following formula: q qI œ spectrophotometric SPF CF EE AAbs ƫ ƫ (2 ) where CF = correction factor (10), EE (λ) = erythema action spectrum (CIE 1987), I (λ) = spectral irradiance received from the UV source, and Abs (λ) = spectrophotometric absor- bance values at wavelength λ. The EE × I values are constants and were determined by Sayre et al. (25).
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