Gong, X.

Knight, J.

Reeves, J.

Tamburic, Slobodanka

Williams, Stefanie

English

UAL Research Online

In Vitro Assessment of Antioxidant Properties of Cocoa Extract in Cosmetic Formulation: Comparison between Analysis by Emitted Light (ABEL) & ORAC Antioxidant Capacity MethodsIntroductionIt is difficult to precisely measure the ability of anti-oxidant ingredients and products to scavenge free radicals. This ability is distinct from the concentration of individual ingredients with claimed antioxidant properties, i.e. the sum of the individual antioxidants may not match the total antioxidant activity of the sample [1]. ORAC (Oxygen radical absorbance capacity) method  is most commonly used to test cosmetics and foods for anti-oxidant capacity [2,3]. However, the accuracy and precision of the ORAC method, especially for materials with strong antioxidant activity, is very poor and not useful for measuring shelf life or for measuring batch-to-batch consistency. ABEL (Analysis by emitted light) antioxidant assays use Pholasin - a protein that emits light in the presence of free radicals and other non-radical ROS [4] . In the series of assays, test material is challenged by a range of different ROS. The result can be compared to antioxidant standards, or presented as an ABEL relative antioxidant capacity (ABEL-RAC) score [5].Tamburic, Slobodanka1; Knight, Jan2; Williams, Stefanie 1,3; Reeves, James2 ; Gong, Xiaoli 21 School of Management and Science, London College of Fashion, London, UK; 2 Knight Scientific Ltd., Plymouth, UK; 3 European Dermatology London, London, UKAimThis study aims to evaluate the novel ABEL-RACmethod for quantifying the antioxidant potential of skincareingredients and products, and to compare the accuracy andreliability of this method to the more established ORACmethod.Material and MethodsMaterialsHigh polyphenol-content Cocoa extractwas obtained from Theobroma cacao beans using special manufacturing procedure (Barry Callebaut, Belgium). Cream formulation containing propylene glycol, carbomer, sorbitanstearate & sucrose cocoate, isopropyl myristate, dicaprylylcarbonate, triethanolamine and preserved water was formulated as a vehicle for Cocoa extract (placebo cream). Two test creams were prepared, with 0.75% and 3.00% of Cocoa extract (CE), respectively.ABEL antioxidant test kits with Pholasin using halogenated oxidants, peroxynitrite and enzyme-generated superoxide, respectively, were supplied by Knight Scientific Ltd, UK. For the ORAC assay all chemicals were purchased from Sigma, UK. For antioxidant analysis, all reagents and standards were freshly prepared at the beginning of each testing day.MethodsThe ORAC assay was performed according to the Fluorescent Microplate Based ORAC Assay Kit instructions obtained from Oxford Biomedical Research, USA.The area under the fluorescence curve was used to quantify the antioxidant capacity of test sample by comparison to a standard curve obtained using a range of concentrations of a water soluble vitamin E analogue, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) The Troloxstandards used were 12.5, 25, 50, 100, 200 µM (working concentrations). The ORAC score was derived from the blank-corrected linear regression curve for Trolox, and units expressed as µmoles Trolox equivalent units per gram of raw material (µmol TE/g).  Figure 1 illustrates a typical response of Trolox standards in the ORAC assay.ConclusionABEL antioxidant method has proven to be of great value to the antioxidant testing of cosmetic ingredients and products. It generates a wide range of relevant data and shows superior reproducibility in comparison with the ORAC method. The ABEL-RAC scores per mg of test material are easy to understand and enable comparisons to be readily made between different materials, batches and other products. The method is particularly useful in testing ingredients of natural origin and resulting formulations.References[1] Knight, J., Knight, R., Quality assurance of nutraceutical health claims: The Case for Antioxidants  Bioworld Europe 4 (2005) 10-13. [2] Prior, R.L., Cao, G., In vivo total antioxidant capacity: comparison of different analytical methods, Free Rad. Biol.& Med. 27 (1999) 1173-1181.[3] Cao, G., Sofic, E., Prior, R.L., Antioxidant capacity of tea and common vegetables, J. Agric. Food Chem. 44 (1996) 3426-3431.[4] Knight, J., Ganderton, M., Armstrong, K., Larkins, N., The use of Pholasin -based assays to evaluate anti- and pro-oxidant capacity of extracts of certain functional foods: the effect of these foods on leucocytes in blood, Free Rad. Biol. & Med 35 (2003), S39[5] Knight, J., Pholasin - based antioxidant assays for cosmetics, cosmeceutical and nutraceutical product development, in Johnson, C. and Loosemore, G. (Eds.) Cosmetic Science Technology, T4 Group, London, UK, 2005, pp. 249-257. +Table I. Summary of ORAC scores for Cocoa extract, tested in 3 independent experiments and at 4 concentrationsIn contrast to the ORAC method, ABEL-RAC scores are not derived from comparison to an antioxidant standard such as Trolox, thus eliminating the assumption that all samples behave the same as a single standard. Secondly, they are not calculated from a single sample concentration; instead a range of concentrations is tested to get a single score. Figure 1. Typical analysis of Trolox standards in the ORAC assay. The area under the fluorescence curve is used to quantify the antioxidant capacity of a sample by comparison with the Trolox standards.The ABEL assays were performed using a BMG Lumistar Galaxy microplate reader, equipped with on-board injectors (BMG Labtech, Aylesbury, UK) and flat-bottomed, white 96-well microplates (Greiner Bio-One Ltd., UK). The final assay volume in each microplate well was always 200 µL.AcknowledgementPholasin and ABEL are registered trademarks of Knight Scientific Ltd. Figure 2. Effective concentration of the test material (normalized to g/L or mg/mL) that reduces the light produced by Pholasin and ROS by 50% (EC50 ); EC50 is used to calculate ABEL-RAC scores.ORAC Score(µmol Trolox Equivalent per g)Test Concentration of Cocoa Extract (µg/mL)0.16 0.31 0.63 1.25MeanConc.CV (%)Assay 1 1290 11258 11306 9395 8312 57.3Assay 2 1896 9532 8715 8653 7199 49.4Assay 3 4284 12513 12256 9717 9692 39.4Mean of Assays 2490 11101 10759 9255CV (%) 63.6 13.5 17.0 5.9ABEL-RAC Peroxynitrite Assay per mg Cocoa ExtractDilution Ranges: µg/mL1,2,4,8 0.75,1.5,3,6 1.25,2.5,1,101 2 3 Mean CV (%)Assay 1 581434 595830 609219 595494 2.3Assay 2 601339 596354 583463 593719 1.6Assay 3 569583 592036 598951 586857 2.6Mean of Assays 584119 594740 597211CV (%) 2.7 0.4 2.2Table II. Summary of ABEL-RAC scores for Cocoa extract, tested in 3 independent experiments in 4 ranges of concentrations Figure 3. Light response curves for a range of concentrations of Cocoa extract (dissolved in propylene glycol) tested in the ABEL antioxidant assay using peroxynitrite. Peroxynitrite is formed in the assay by the reaction between superoxide anion and nitric oxide, released simultaneously and continually from 3-morpholino-sydnonimine hydrochloride (SIN-1), injected at time zero.In the second part of the study, a range of cosmetic formulations and ingredients were tested in three ABEL antioxidant assays to monitor the effect of CE incorporation (at different levels) in cosmetic creams, and to monitor their shelf life over 5 weeks. For all three ROS challenges, the addition of CE (0.75 and 3 %) to the emulsion cream resulted in a measurable dose-response increase in antioxidant capacity of the creams. The calculation of the predicted ABEL-RAC scores was based on the ABEL-RAC score of the pure CE and its inclusion level in each cream.  An example of the results can be seen in Table III.  Three  types of ABEL antioxidant assays were performed:•with halogenated oxidants•with peroxynitrite •with enzyme-generated superoxideCalculation of ABEL-RAC scoresSeparate light response curves were produced for each concentration of a material tested, as well as for solvent control. The EC50 (effective concentration ) is the concentration (normalized to mg/mL) of a material that reduces the light produced with Pholasin and the free radical or other ROS by 50% (Figure 2). The greater the amount of material required to reduce the light by half, the weaker the antioxidant capacity. The EC50 values are converted to positive relative antioxidant capacity scores (ABEL-RAC mg scores) for each free radical or oxidant used to challenge the test material. ABEL-RAC mg scores are the reciprocal of the EC50 values multiplied by 100. Therefore, the higher the ABEL-RAC mg, the higher the antioxidant capacity of the sample.  Results and DiscussionVery high antioxidant capacity scores of the Cocoa extract were measured with both ORAC and ABEL-RAC methods (Tables I and II), which is in agreement with its published ORAC values of 11,000 µmol TE/g (Barry Callebaut, Belgium). It is known that the ORAC method uses the net area under the curve (AUC) of one concentration of a test sample to calculate the ORAC score, by comparison to a Trolox standard curve [3]. To assess ORAC intra-assay reproducibility, we calculated ORAC scores using different concentrations of the same sample in the same assay. Table I shows that the ORAC scores (per gram) of CE were highly dependent on the concentration used for testing. The biggest variation in the ORAC score in a single assay produced a range between 1290 and 11306 µmol TE/g, resulting in coefficient of variation (CV) value of 57.3% for the four different concentrations tested. The differences in ORAC scores may be due to the fact that the net AUC is not linearly related to the sample concentration as it is with Trolox.This eliminates variability that may be introduced when calculating scores using a single point calculation, as shown in the ORAC assay. It also means that even when different concentration ranges are used, ABEL-RAC scores are highly reproducible, with CV values < 3% between ranges (Table II). Simalar findings were obtained when day-to-day reproducibility of ORAC and ABEL-RAC scores were assessed (Tables I and II).SampleSOD EU (predicted)SOD EU  (actual)Synergy (%)Propylene glycol - antioxidant -Cocoa extract - 396000 -0.75% Cream (Fresh) 2970 2975 1000.75% Cream (5 weeks 40C) 2970 1600 540.75% Cream (5 weeks RT) 2970 2175 733% Cream (Fresh) 11880 13250 1123% Cream (5 weeks 40C) 11880 10625 893% Cream (5 weeks RT) 11880 11625 98Table III. Superoxide dismutase equivalent units (SOD EU) of Cocoa extract and cream formulations containing Cocoa extract as evaluated using superoxide (enzyme-generated). Predicted SOD EU values are based on the SOD EU score of the pure Cocoa extract and the inclusion level (w/w) of the Cocoa extract in the cream formulations. The % synergy is the actual SOD EU score represented as a percentage of the predicted SOD EU score. 0 15000 30000 45000 0 Time (seconds) Light --- Control sample (maximum light of Pholasin& peroxynitrite without competition fromantioxidant)--- A sample assayed at its EC50 concentrationEC50

In vitro assessment of antioxidant properties of cocoa extract in cosmetic formulations: comparison between analysis by emitted light (ABEL) and ORAC antioxidant capacity methods

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In vitro assessment of antioxidant properties of cocoa extract in cosmetic formulations: comparison between analysis by emitted light (ABEL) and ORAC antioxidant capacity methods

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