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A Non-Invasive Method for the in Vivo Determination of Skin Antioxidant Capacity

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  A non-invasive method for the  in vivo  determinationof skin antioxidant capacity (IAC-S s ) Paola Ziosi 1 , Elena Besco 2 , Silvia Vertuani 2 , Nicola Solaroli 1,3 and Stefano Manfredini 1 1 Dipartimento di Scienze Farmaceutiche, Universita` degli studi di Ferrara, Ferrara, Italy,  2  AmbrosiaLab s.r.l., Via Fossato di Mortara 17/19, Ferrara, Italy and  3 Division of Metabolic Diseases, Karolinska University Hospital, Stockholm, Sweden Background/purposes:  Skin antioxidant network protectscells against oxidative injury and prevents the production ofoxidation products. When oxidative stress overwhelms theskin antioxidant capacity, the subsequent modification of thecellular redox apparatus leads to an alteration of cell home-ostasis leading to degenerative processes. In the dermo-cosmetic field, the topical application of antioxidants is oftensuggested as a possible strategy to prevent and modulateoxidative skin damages. Continuing our studies addressedto set-up new bio-engeneering protocols for the claim sub-stantiation of antioxidant cosmetic products, we have devel-oped a new non-invasive methodology for the evaluation ofantioxidants cosmetics ingredients and finished products. Methods:  The effects of a pre-treatment on forearm skinwith an antioxidant ingredient were investigated on 15volunteers, in a double-blind randomised fashion. A non-invasive method was devized that comprises the collectionof forehead SC layers of the pre-treated area and controland the next evaluation of skin antioxidant capacity (IAC-S s ) by a luminescence-based method. Results:  The results showed that the antioxidant prepara-tion was able to increase, to a statistically significant extent( P  o 0.01), the IAC-S s in comparison with the controlarea. The data were confirmed ( P  o 0.05) by comparisonwith a method, previously developed by us, based onDermAnalyzer s . Conclusions:  In view of the simplicity and reliability of themethod here presented, this new technique is proposed as apossible tool for the routine evaluation of  in vivo   efficacy ofantioxidant functional ingredients and finished products. Key words:  photochemiluminescence – skin antioxidantcapacity – Dermanalyzer s – tocopherol &  Blackwell Munksgaard, 2006 Accepted for publication 3 January 2006  S KIN IS  a highly metabolic tissue that possessesthe largest surface area in the body, and isalso a major candidate and target of damagingfree radicals. It is well-known that free radicalsand reactive oxygen species (ROS) are involvedin the mechanism leading to cutaneous damages,such as early ageing, inflammatory disorders andskin cancers. The skin can be protected fromoxidative damage with elaborate and diversifiedantioxidant mechanisms, of enzymatic and non-enzymatic nature. Antioxidants, such as  a -toco-pherol, ascorbic acid, and other active substanceswith high radical scavenging activity, modulatethis damage by scavenging free radicals and lipidperoxyl radicals. An important goal of moderncosmetic science should be the development of appropriate skin care products based on topicalapplication of radical scavengers to prevent theonset and propagation of the free radical cascade.Although many studies strongly suggest thatsome cosmetic ingredients, such as vitamins,minerals and botanicals, have the potential toimprove skin ageing, rigorous studies, that canwithstand critical review, are still lacking in thisfield, as most of the studies conducted are eithersmall in scale,  in vitro  or utilise non-humanmodels.After its description by Pinkus (1), tape strip-ping has become a standard method in dermato-logical research (2). Tape stripping is a relativelynon-invasive technique, which permits samplesof the stratum corneum to be collected from theskin. Taking this into account and continuing ourstudies (3) addressed to set up new bio-engeneer-ing protocols for the claim substantiation of anti-oxidant cosmetic products, we have developed anon-invasive and quantitative method that canreliably measure the integral skin antioxidantcapacity (IAC-S s , AmbrosiaLab sri, Ferrara,Italy), by means of the tape stripping technique 303 Skin Research and Technology 2006;  12 : 303–308  &  2006 The Authors Journal compilation  &  2006 Blackwell Munksgaard 2006 Printed in Singapore   All rights reserved Skin Research and Technology  coupled to an efficient method for antioxidantactivity determination (4). In particular, in thisstudy we have validated our approach using asimple vitamin E-based formula to modify theIAC-S s of the stratum corneum. To this end, theantioxidant capacity of the stratum corneum wasdetermined by a photo-chemiluminescence(PCL)-based method and compared with thatobtained after a pre-treatment with the studyantioxidant formula. Materials and Methods  Antioxidant formula In order to reduce the number of parameters, theformula was limited to the simplest possible andthus to the lowest number of ingredients andexcipients. The antioxidant ingredient was cho-sen from the panel of ingredients available at ourlaboratory on the basis of the highest antioxidantactivity and lowest skin irritation. The chosenformula contained (according to INCI terminol-ogy): cyclometicone (Polichimica, Bologna, Italy)and tocopherol (Copherol F1300, Cognis, CareChemicals, Fino Mornasco, Como, Italy). Ratioof the assay mixture was 1:1.A standardized patch test of the mixture wascarried out. The test involves the application of the examined substance to the skin under adhe-sive tape, which is then left in place for 48h. Theskin is then examined a further 48h later for anyresponse. The results demonstrated that topicalapplication of the antioxidant mixture is notassociated with acute skin irritation or withallergic sensitization. Subject and experimental design: in vivo study Fifteen Caucasian healthy volunteers between 23and 30 yearsparticipated in thestudy aftergivingwritten consent. The study was approved by theEthics Committee. The study was carried out in apartly air-conditioned room at a temperature of 23    2  1 C and with an average relative humidityof 33%. The study was performed in a double- blind and randomized manner. The anatomicalsites chosen were four areas (10cm 2 ) of theinternal forearm. Two areas (A and B) weretreated with the product, whereas C and Dwere control areas. The study was performed intwo steps. The first was the pre-treatment phaseconsisting of the application of 20 m L of theantioxidant mixture onto skin areas of the humanvolunteers twice a day for 15 days. The secondphase, starting from day 16, involved the evalua-tion of the effects of the pre-treatment on theforearm skin of the 15 volunteers, using two non-invasive techniques: tape stripping/PCL assay incomparison with Dermanalyzer s (AmbrosiaLabsri, Ferrara, Italy). Tape stripping The areas of the forearm were marked andrectangles of exactly fitting pieces of adhesivetape (cut to a size of 2cm    5cm) were consecu-tively applied to the skin.Before sample acquisition, the skin wascleaned with a piece of gauze soaked in 50 m L of ethanol. The stratum corneum of the treated areawas removed by 11 successive tape strippingsusing ‘3M’(3M,Minneapolis,MN,USA) invisibleadhesive tape. The tape was applied to the testsite with forceps, flattened equally twice, andremoved gently using moderate and even trac-tion. Owing to the possibility of surface contam-ination and to remove surface lipids, the first(uppermost) tape stripping was discarded. Sub-sequent tapes were then placed, adhesive sidedownwards, into a glass box with 15mL of MeOH (HPLC grade) and then treated for30min in an ultrasonic bath. Solutions werepurified by centrifugation (10min with 4000cy-cles/s) to separate small horny layer particles,and the uppermost part was analysed.  Antioxidant capacity Measurements of antioxidant capacity of thetocopherol formula and stratum corneum sam-ples were performed by photochemilumines-cence assay (PCL).Skin antioxidant capacity of volunteers wasmeasured at baseline and after 15 days (at theend of the treatment period). The method princi-ple (PCL) is briefly described: defined free radi-cals (superoxide anion radicals) are generated inthe measuring system by the exposure of aphotosensitizer to a UV-light source. The freeradicals are detected by their reaction with achemiluminogenic substance and the measure-ment of the emitted light. The light flashes aredetected by a photomultiplier. These generatedradicals are partially scavenged by reaction withthe sample antioxidants and remaining radicalsare quantified by the above-described detec-tion principle. The results are presented in 304 Ziosi et al.  equivalent concentration units of Trolox s (Sigma-Aldrich, St. Louis, MI, USA) (synthetic vitamin E)for lipid-soluble substances or ascorbic acid forwater-soluble substances. Different concentra-tions of these standard compounds are used toestablish a calibration curve, and the detectorsignal of each run is monitored for 180s. ThePCL assay is suitable to measure the radicalscavenging properties of single antioxidants aswell as more complex systems in the nanomolarerange (5). The antioxidant potential is measured by means of the lag phase at different concentra-tions, calculated by a Trolox s calibration curveand expressed as mmol equivalents in antioxi-dant activity of a reference compound (i.e.Trolox s ). The PCL method was carried out asdescribed by Popov and Lewin (6, 7) and, asdecribed above, can be conducted by two differ-ent protocols, ACW and ACL, that consent tomeasure the antioxidant capacity of the water-and lipid-soluble components, respectively. Inthe water-soluble fraction antioxidants such areflavonoids, ascorbic acid, aminoacids, etc. aredetected, while in the lipid-soluble fraction, toco-pherols, tocotrienols, carotenoids, etc. are mea-sured.  ACW and ACL sample preparation – generalprocedure An exact quantity of the product was poured in1mL methanol HPLC grade, for the measurewith the ACL kit, or 1mL water, HPLC grade,for the measure with the ACW kit, and they weremixed by vortex for 1min at room temperature.The obtained solution was then filteredthrough HPLC filter (Chemtek Analitica, Bo-logna, Italy) by a syringe and diluted with Re-agent 1 of ACL or ACW kit (AnalytikJena, Jena,Germany). Different dilutions of the varioussamples were evaluated to obtain a calibrationcurve.Results are expressed as mmol equivalents, inantioxidant activity, of Trolox s for each litre of product under examination. DermAnalyzer s The data obtained with the protocol describedabove, were compared with those collected by amethod, previously developed by us, based onDermAnalyzer s .In thismethod, theintensity andduration of skin redness generated by methylnicotinate (MN) is used to assess the efficacy of functional ingredients, included in cosmetic for-mulations to prevent and modulate skin oxida-tive damages (3). The reliability of the method inthe measurement of skin redness has been pre-viously assessed by us in comparison with otherwell-recognized instruments. In particular, thecorrelation with Minolta Chromameter CR300(Minolta, Tokyo, Japan) was very high both  invitro  and  in vivo  ( R 5 0.99 and 0.86, respectively)(3). We used the standardized protocol as de-scribed before (3). Briefly, after the pre-tratmentphase, consisting of the application of the pro-duct studied twice a day for 15 days, the selectedareas were re-treated with the products, 30 0  before the application of the irritant agent. Atthis time the baseline value of redness of thetreated and control areas (T0) was determined.The irritation was induced in both areas by theapplication of paper disks (5mm of diameter)soaked with 15 m L of 0.5% aqueous solution of methyl nicotinate (99% purity, Fluka ChemieGmbH, Sigma-Aldrich, St. Louis, MI, USA) for3 0 . After removal of the filter disk, excess solutionwas gently removed using a paper tissue. Acircular erythema spot was induced by this treat-ment. Measurements were performed before MNexposure (T0) and 15min after MN removal.  Image recording In order to develop a simple and cost-effectivemethod, a simple digitalizer was chosen andcoupled with specifically developed softwaredescribed below. True colour images of the ob-served skin area were taken under the sameconstant lighting conditions, using a digitalphotocamera (Nikon coolpix4300, Nikon, Tokyo, Japan) placed over a specifically designed dark box. The position of the camera was adjusted sothat the examined areas were in the middle of theimages. In particular, great attention has beengiven to the type and modality of illuminationand in consequence to white calibration. Thecamera was used in manual mode and eachparameter was accurately defined. In particular,for illumination, a diffuse light (D-65, 6500 Kel-vin, Osram GmbH, Berlin, Germany), which isconsidered a standard for average daylight, witha minor yellow component, was used to reduceshadows on the border of the arm and to avoidreflections on the skin. The size of the skin areaunder study was 10cm 2 , and it was located onthe volar forearm. The arm was placed inside the 305 In vivo  determination of skin antioxidant capacity (IAC-S s )   box, volar forearm upwards, and fixed to avoidlarge movements at suitable distances and anglefrom the camera and light source. To analyse thedegree of the erythema response, true colourimages were taken at baseline and 15min afterMN removal (T0), which represents the highestpeak of redness. The reliability of such a devicehas been already demonstrated by us during aprevious comparative study (3). Software The images were downloaded from the digitalcamera and archived on the computer hard diskand afterwards analysed singularly with theDermAnalyzer s . The program was selected eacharea to be treated simply by drawing a linearound the zone to be analysed. After this step,the program algorithm, developed by us, auto-matically pulls apart the treated skin from theuntreated skin, and measures the CIE  a*   compo-nents of the red area, excluding the other colourcomponents such as  b*   (green–yellow). The finalresult consists in the mean  a*   value of the wholearea selected. The reliability of such a programhas been already demonstrated by us during aprevious comparative study (3). Erythema–pruritus–redness s index The self-pruritus sensation and the degree of oedema after MN removal was also determined.In this test the volunteers were asked to score theself-pruritus sensation induced by topical MNapplication at the same time skin erythema wasdetermined by DermAnalyzer s . The simple scor-ing system is as follows: 0, no pruritus; 0.5, weakpruritus sensation; 1, moderate pruritus sensa-tion; 2, high pruritus sensation; 3, intolerable.Moreover, erythema and oedema were scoredvisually using the following scale: 0, no reaction(negative); 0.5, very weak erythema or minutescaling (doubtful); 1, weak erythema, slight oe-dema, slight scaling and/or slight roughness(weak); 2, moderate degree of erythema, oedema,scaling and/or roughness, or minor degree of erosion, vesicles, bullae, crusting and/or fissur-ing (moderate); 3, marked degree of erythema,oedema, scaling, roughness, vesicles, bullae,crusting and/or fissuring (strong); 4, as 3, withnecrotic area (very strong/caustic).According to that we have introduced a newparametertermedEPR s indexwhichrepresentsthesum of the three parameters: redness, measured byDermAnalyzer s , degree of erythema/oedemascored visually and self-pruritus sensation. Statistical analysis For both test mean values and standard devia-tions were calculated using the software programGraph Pad prism. Paired one-tailed Student’s t -test was used in comparison with significancedifference between the treated and control area.A significance of   P o 0.05 was used as the valuefor significant differences. Results The antioxidant capacity of the 1:1 tocopherolformula was 1540mmol Trolox s equivalents/L.This result is quite consistent and may be as-cribed to the natural vitamin E preparation usedfor the formula.  Measurements of IAC-S s Results are summarized in Table 1 and representsthe sum of ACW and ACL mean values bothexpressed in Trolox s equivalents per litre of solution. In order to evaluate skin antioxidantcapacity, 10 sequentially tape-stripped layers wasobtained from each volunteer. Measurementswere taken in triplicate and overall mean valuesfor the 15 volunteers were calculated. Datashowed that IAC-S s was significantly higher inpre-treated areas with respect to the control( P o 0.01). In fact, whereas the value of area Aincrease from 24.76 m mol Trolox s /L (T0) to36.86 m mol Trolox s /L (T15), the control areashows no difference between T0 and T15 (theend of the treatment period) (Fig. 1).  Measurements of EPR s index Mean values among the 15 volunteers and thestandard error of measurements (SEM) for EPR s index are shown in Table 2. As it can be observed(Fig. 2) the antioxidants formulation tested wasable to reduce, in statistically significant extent,the intensity of skin redness induced by MN TABLE1. Mean of values of IAC-S s expressed as  m mol Trolox/L andstandard deviation (n 5 15) at baseline (T0) and after the pre-treatmentwith antioxidant formulation (T15) T0 T15 % variationArea A 24.76 (  1.3) 36.86 (  1.6) 48.86Control 25.19 (  1.2) 25.21 (  1.4) 0.08 306 Ziosi et al.
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