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A test for antioxidant activity in cosmetic formulations

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The aim of this study was to develop a technique to assay for the activity of antioxidants in a finished cosmetic product. This was accomplished by adapting the Randox Assay for Total Antioxidant Status kit so that diluted samples could be evaluated
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  j. Cosmet. ci., 53, 237-240 (July/August 2002) A test for antioxidant ctivity n cosmetic ormulations E. PELLE, T. MAMMONE, K. MARENUS, D. DICANIO, and D. MAES, Estee auder Research aboratories, 25 Pindawn Road, Me/ville, NY l 1747. Accepted or publication arch 15, 2002. Synopsis The aim of this study was to develop a technique o assay or the activity of antioxidants n a finished cosmetic roduct. This was accomplished y adapting he Randox Assay or Total Antioxidant Status kit so that diluted samples ould be evaluated y kinetic as well as end-point determinations. sing this technique, we found hat a finished roduct had an IC5o of 0.07 gm of product and a relative antioxidant activity concentration f 52.7 nmoles/mg. INTRODUCTION Environmental nsult to human skin by ultraviolet UV) radiation, as well as by cigarette smoke nd air pollution, generates eactive xygen ntermediates hat contribute o both acute and chronic skin damage 1,2). For example, mmediately after overexposure o sunlight, an erythemal esponse s induced hat is associated ith epidermal nflamma- tory oxidative reactions. Moreover, n terms of chronic exposure, he involvement of oxygen ree radicals as also been mplicated n actinic skin damage hat manifests tself in elastosis, ollagen isorganization, nd most notably n the appearance f wrinkles 3). Due to increased outdoor leisure activities, these visible signs of photodamage nd premature aging have become widespread n our society. To address his problem, the cosmetics ndustry has devoted much research oward the development of various skin care products. Although protective sunscreen roducts hat absorb UV and diffuse photonic energy are widely used, cosmetic roducts hat contain antioxidants, which scavenge deleterious eactive oxygen species produced in skin after environmental trauma, have also become standard or a healthy skin care regimen. Although analytical echniques re available o measure he level of antioxidants n cosmetic products, n general, they do not provide any information regarding their potential activity. Further, due to the complex nature of cosmetic ormulations, xtract- ing and determining biochemical ctivity in a finished product can be a challenging task. Previously, we evaluated he antioxidant potential of certain cosmetic ngredients (4) and also he antioxidant efficacy of finished products on skin (5). In this study, we 237  238 JOURNAL OF COSMETIC SCIENCE now report on a novel echnique o measure ntioxidant activity directly in a complete cosmetic product. MATERIALS AND METHODS SAMPLE PREPARATION A typical cosmetic ormulation ontaining blend of emulsifiers as prepared s either a control with no antioxidants r as a complete ormula with a mixture of antioxidants. The following antioxidants were used in the formulation: 2.0 tocopheryl acetate (Hoffman-LaRoche, arsippany, J), 0.1 butylated hydroxy oluene Rhone-Poulenc, Cranbury, NJ), 1.0 magnesium scorbyl hosphate Barnet, Englewood, NJ), 0.1 ubiquinone 50 and 0.5 N-acetyl-L-cysteine Seizer, Carlsbad, CA), 0.1 rosemary (Robertet, Oakland, CA), and 0.1 tocopherol ysteamine Mercier, S. Plainfield, NJ). ASSAY The Randox Assay or Total Antioxidant Status kit (Randox, Antrim, UK) was adapted for use n cosmetic roducts y diluting the formulations o be tested o 1 in isopropyl alcohol. At 1 in isopropyl alcohol, the samples are sufficiently clarified and the antioxidants olubilized o allow the reaction o proceed without interference. riefly, 2,2'-azino-di-(3-ethylbenzthiazoline ulphonate) ATBS) is reacted with a peroxidase and H20 2 to convert ATBS into a radical cation. n this state, ATBS forms a chromogen that can be measured pectrophotometrically t 600 nm. In the presence f antioxidants, this color ormation s inhibited. Typically, 50-100 pl of the 1 sample s diluted in water up to 250 pl. Then, 1.5 ml of chromogen olution s added, ollowed by the addition of 0.3 ml of substrate solution. The absorbance A) of the samples s then measured mmediately n a Beckman DU-7500 spectrophotometer sing the kinetics/ time program. CALCULATIONS Percent nhibition as calculated s dAvehicle-dAproduct/dA,•ehicle) 100 and used o quantitate n C5o value. Also, a range f 15 to 85 nanomoles f an antioxidant tandard (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic cid) was used o determine he relative activity of a product. RESULTS One hundred microliters 1 mg) of a 1 cosmetic ample dilution was assayed, nd a typical three-minute inetic plot of the data s shown n Figure 1. The sample without antioxidants ad a dA/min of 0.126 whereas he sample ontaining ntioxidants ad a dA/min of 0.011. Thus, there was an 86.8 decrease rom the control sample. From similar kinetic plots, he average alue hat would nhibit the reaction y 50 was hen determined o be 0.7 mg of the sample. Since the sample was a 1 dilution of the  TEST FOR ANTIOXIDANT ACTIVITY 239 Zoom ZoomOut Trace fluLoscale flnnotate Print 8.58088 [Abs] 8.88888 8.8888 ........................... : .......................... : .......................... : ........................... • .......................... : : sec 188.88 Figure 1. Kinetic plot of the increase n absorbance t 600 nm over ime. (-I•-): Vehicle ormulation was 0.126 dA/min. (-(2)-): Antioxidant formulation was 0.011 dA/min. product, 0.7 mg is multiplied by 100 in order o determine hat 0.07 gm of the cosmetic product s equivalent o 50 inhibition in this assay. n this way, relative measurements of effectiveness an be calculated nd used or comparison o other products. Based on regression nalysis rom the standard urve and end-point assay nalysis, he relative antioxidant activity concentration hat was determined or this product was 52.7 nmoles/mg + SD 1.7) of material. n contrast he placebo ontained nly 11.4 nmoles/ mg (+ SD 1.2), although when compared o a blank, there appeared o be some background ntioxidant ctivity by the formulation tself. These data are summarized n Figure 2. 40 20 lO blank vehicle vehicle + antioxidants Figure 2. Increase n the amount of nmoles/mg f antioxidant activity n cosmetic ormulations s deter- mined by standard curve and end-point assay measurements.  240 JOURNAL OF COSMETIC SCIENCE DISCUSSION Antioxidants n skin care products have been ound to be effective protectants gainst free radical-mediated xidative amage n skin. Also, due to the rise n photodamage n an aging population, he topical application of cosmetic roducts hat contain antioxi- dants has become n important area of research n skin care products. Thus, the need o measure he activity of antioxidants n a finished product s of critical mportance. n this report we describe novel and simple echnique o quickly assess he relative antioxidant potential of whole product ormulations. Additionally, it can be utilized for stability studies nd, since he chromogen evelops n the visible region, perhaps ven a quali- tative result can be obtained by workers n the field who lack spectrophotometers ut need to assay he antioxidant activity of a product. REFERENCES (1) B. A. Gilchrest, Skin and Aging Processes CRC Press, Boca Raton, FL, 1989), pp. 97-116. (2) A. V. Benedetto, he environment nd skin aging, Clin. Dermatol., 6, 129-139 (1998). (3) J. Fuchs and L. Packer, Eds., Oxidative tress n Dermatology Marcel Dekker, New York, 1993). (4) E. Pelle, D. Maes, G.A. Padulo, E-K. Kim, and W. P. Smith, An in vitro model to test relative antioxidant potential: Ultraviolet-induced ipid peroxidation n liposomes, rch. Blochem. iophys., 283, 234-24O (1990). (5) E. Pelle, N. Muizzuddin, T. Mammone, K. Marenus, nd D. Maes, Protection gainst ndogenous nd UVB-induced oxidative damage n stratum corneum ipids by an antioxidant-containing osmetic formulation, Photodermatol. hotoimm•nol. hotoreed., 5, 115-119 (1999).
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