MAGAZINE. A Psychophysiological Approach to Substantiate Efficacy of Bath Additives From an Eco-Friendly To an Eco-Improving World of Cosmetic Science

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1 Volume 178 Number 3 October 2015 MAGAZINE The Global Publication of the International Federation of Societies of Cosmetic Chemists Investigation of Consumers Hair Shine Perception by Eye Tracking Technology in Combination with Assessment of Physiological Body Reactions Decorative Cosmetics: In Vivo Facial Measurement of Color Parameters, Even Skin Tone and Radiance Swertia chirata Extract Promotes Epidermal Regeneration through Stimulation of Paracrine Communication between Adipose-Derived Stem Cells and the Epidermis A Psychophysiological Approach to Substantiate Efficacy of Bath Additives From an Eco-Friendly To an Eco-Improving World of Cosmetic Science

2 Scientific Papers Investigation of Consumers Hair Shine Perception by Eye Tracking Technology in Combination with Assessment of Physiological Body Reactions Wolf Eisfeld, Daniela Prinz, Björn Schröder, Jennifer Schmidt, Ralf Stürmer 3 Decorative Cosmetics: In Vivo Facial Measurement of Color Parameters, Even Skin Tone and Radiance Stephan Bielfeldt, Marianne Brandt, Nathalie Lunau, Matthias Seise, Gunja Springmann, Klaus-P. Wilhelm 11 Swertia chirata Extract Promotes Epidermal Regeneration through Stimulation of Paracrine Communication between Adipose-Derived Stem Cells and the Epidermis Carine Bézivin, Arléty Pérolat, Estelle Loing 17 A Psychophysiological Approach to Substantiate Efficacy of Bath Additives Ralf Stürmer, Jürgen Blaak, Mareile Opwis, Jennifer Schmidt, Peter Staib, Rainer Wohlfart, Wolfram Boucsein 23 From an Eco-Friendly To an Eco-Improving World of Cosmetic Science Addressing Water Pollution in Emerging and Developing Societies Chaikriangkrai Amata 31 IFSCC Magazine: Official scientific magazine of the International Federation of Societies of Cosmetic Chemists Publications Chair: Andrea Weber, Germany Chair Science Committee: Fujihiro Kanda, Japan Chair Education Committee: Miki Minaminu, Japan Scientific Editor: Lothar Motitschke, Germany Language Editor: Marcia Franzen-Hintze, Germany Honorary Auditors: Anne Hunt, France Judy Beerling, United Kingdom 4 issues / year Copyright 2015 Arranged in Germany ISSN# IFSCC Office/Publisher s Office IFSCC Suite 109, Christchurch House 40 Upper George Street Luton, Beds LU1 2RS, UK Tel: Fax: lorna org Front Cover Design: Fabian Mai fabian com Layout: Andrea Weber Important Information From 2014 onwards we publish the contents of the IFSCC Magazine on the IFSCC Website: www ifscc org You will have to sign in to the Member Zone where you can then find the Scientific Papers of the IFSCC Magazine Detail to be found on the Website You will find the contents on the IFSCC Website as follows: News from Member Societies: News Page on Website Awards: Awards Pages on Website Educational Programs: Education Pages on Website Scientific Papers: Member Zone 2 IFSCC Magazine

3 Investigation of Consumers Hair Shine Perception by Eye Tracking Technology in Combination with Assessment of Physiological Body Reactions Wolf Eisfeld 1, Daniela Prinz 1, Björn Schröder 1, Jennifer Schmidt 2, Ralf Stürmer 2,3 1 BASF Personal Care and Nutrition GmbH, Henkelstr. 67, Düsseldorf, Germany 2 Psyrecon research & consulting, Institut für angewandte Psychophysiologie GmbH, Düppeler Str. 38, Wuppertal, Germany 3 FOM Hochschule für Oekonomie und Management, Herkulesstraße 32, Essen, Germany This publication was presented as a podium presentation at the 28. IFSCC Congress, October 27-30, 2014, Paris, France. Keywords: Hair shine, eye tracking, objective emotional assessment, psychophysiology, order phenomena ABSTRACT INTRODUCTION Shiny hair is one of the most desirable cosmetic attributes [1] [2]. It is also associated with youth and health and consequently elicits positive emotional responses. The enhancement of hair shine is a frequent claim of hair care products. However, the determination of hair shine by physical measurements and visual assessment by a test panel is still a challenge, with especially correlation of the two methods being problematic. Sensory properties of hair shine, such as specular or diffuse reflection, sparkle and luster, are hard to define scientifically. Though it is common knowledge that both absolute intensity of reflected/scattered light as well as the contrast between bright and dark areas on the hair are the main factors for hair shine perception and assessment, there are most probably also other very important factors like hair arrangement (from fiber alignment to overall hairstyle), color (natural and artificial) and dynamics (e.g., traveling of shine band during movement of head) which play an important role and whose relative importance is not known. The goal of this investigation was two-fold: to elucidate fundamental mechanisms of how consumers perceive hair shine of differently The goal of this investigation was to elucidate fundamental mechanisms of how consumers perceive the hair shine of differently treated hair tresses. The spatial and temporal evolution of the visual focus of test persons on the hair tress during the evaluation task was recorded using eye tracking as a novel tool for hair shine evaluation. Simultaneously, the established technique of Objective Emotional Assessment (OEA) was used to record physiological body reactions. The test samples, Chinese hair tresses that had been bleached 1 4 times, were presented to volunteers either separately or pairwise for comparison. The eye tracking data was analyzed with respect to spatial resolution and linked to the emotional response of the test person as inferred from the OEA data in order to investigate the emotional impact of the perceived hair shine on the person. The results indicate that there is a clear separation of the shine properties evoked by the different bleaching treatments. Furthermore this study suggests that the perception of hair shine is linked both to the areas of highest contrast and highest reflection intensity on the presented hair tresses. In addition, there is a strong indication that a comparative presentation of treated hair tresses and to take into account the strong emotional component of this desired property, which so far has been mainly neglected in the existing scientific work. Therefore, two different experimental techniques were combined for the first time. Eye tracking was used as a novel tool for hair shine evaluation to record the spatial and temporal evolution of the visual focus of test persons on the hair tress during the evaluation task and the established technique of objective emotional assessment used simultaneously to record psychophysiological responses. EXPERIMENTAL Eye tracking Eye tracking is a commonly used tool for the evaluation of consumers perception [3] [4]. This technique was deployed already decades ago to track the attention in complex situations such as driving a car and also to monitor the focus of customers while presenting marketing material such as advertising or product placement. In static presentations of content, heat map overlays are an elegant way to visualize the area of interest of the test person both spatially and with respect to duration/time. hair tresses to a test person is biased by positional preferences of the subject. Moreover, order phenomena obviously play an important role in hair shine perception, since slight irregularities like dust or hair fibers sticking out of an ordered tress clearly attracted the attention and gaze fixation of the volunteers. By revealing such a variety of fundamental effects, which all have significant impact on subjective hair shine evaluation, this study shows that combination of the two complementary techniques eye tracking and OEA can be used successfully to further elucidate the perception of hair shine. Although eye tracking has already been used for scientific work especially in the area of skincare and color cosmetics (e.g. [5]-[8]), examples are not very numerous and the application to hair just very recently has appeared in the literature [9] [10]. In this study eye tracking was used to analyze the gaze of volunteers on hair tresses, with a special focus on hair shine properties. Therefore, to produce well differentiated hair samples, Chinese hair tresses (10 cm long, 2 cm wide, International Hair Importers & Products, Glendale/USA) were bleached once, twice, three and four times (bleaching conditions: ultra-bleaching with 6% H 2 O 2, 13% NH 4 S 2 O 8 ), with the untreated dark hair representing a sample with strong specular reflection and low diffuse scattering, and the four bleaching grades representing samples with increasing degrees of diffuse scattering (Figure 1). All five different samples were presented as stimuli to volunteers, either separately or in pairwise comparison. For presentation to the volunteers, the hair tresses were mounted on circular sample holders (Figure 1) and fixed in the custommade box displayed schematically in Figure 2, resulting in a visible tress length of 5.5 cm. An opening on the rear side of the IFSCC Magazine

4 Figure 1: Samples presented as stimuli Chinese hair untreated (A), bleached once (B), twice (R = reference), three times (C), and four times (D). box allowed easy manual exchange of the hair samples during the experiment. A defined and constant source of light was mounted in the upper part of the box, invisible to the volunteer. The eye tracking device was placed below the hair sample holders. To suppress unwanted reflections, the interior of the box was covered with black velvet and a lightproof black cloth was used to cover the box entrance and the volunteer s head. The position of the volunteer s head was fixed at a distance of 50 cm from the hair tresses with the help of a chin rest. The eye-tracking device consisted of a recording element with one camera and two infrared emitters covered by a black glass screen to prevent distraction of the participants. The hardware was manufactured by CanControls (Aachen/Germany). Special high-resolution eye-tracking software (GazeControl) was customized along with the hardware to allow for highly sensitive gaze registration. The software allowed for automatic scanning of the participant s pupils and accurate tracking after an initial individual calibration on a 4x3 grid. The camera and software acquired gazes at a frame rate of 20 Hz. Data was saved as a tabular text file (csv format), including time stamps and x-/y-coordinates of the individual gaze paths per participant and stimulus confrontation. After data aggregation, percentages of views on the stimuli were calculated for each horizontal 1 mm segment of the overall 5.5 cm visible sample length. This procedure was set up to extract the regions on the hair tresses that were most often viewed and therefore account for the most emotion-eliciting areas. Psychophysiological assessment To be able to link these spatially resolved eye-tracking data to the emotional response of the volunteers, thus investigating the emotional impact of the perceived shine on the test person, the emotional response was determined by means of objective emotional assessment. This method was developed to analyze emotional consumer experiences, especially in response to slight differences in product properties (for a review, see [11]). The physiological responses of volunteers, such as electrodermal activity, electrical muscle signals in the face and the heart rate, were recorded while looking at the different hair tress stimuli. Figure 2: Left: Schematic setup of the presentation box with hair tress holder, light source, eye-tracking device and position of volunteer s head. Right, upper part: a view into the presentation box; lower part: a view from the eye-tracking camera. Table I: Psychophysiological parameters used for OEA analysis Psychophysiological recording for objective emotional assessment has been described in detail elsewhere ([12] [13]). In short, biosignals to assess implicit emotional responses were acquired by means of the modular Biopac MP150 System. Besides the main amplifier, modules for recording skin conductance (SC), electrocardiogram (ECG), blood volume pulse (BVP), electroencephalogram (EEG) and activity of three facial muscles via electromyography (EMG) were used with the corresponding sensors and electrode creams (for an overview of the measures and recorded parameters, see Table I). Skin conductance was measured on the palm of participants nondominant hand; the electrocardiogram was recorded on the right clavicle, left lower costal arch and sternum; the blood volume pulse was recorded with a finger sensor on the index finger of the non-dominant hand. For the electromyography, the Zygomaticus, Corrugator and Masseter muscles were monitored and electrodes placed on the middle and end of each facial muscle, respectively. The electroencephalogram was acquired with a unipolar recording setup measuring on the vertex electrode (Cz) with the reference on 4 IFSCC Magazine

5 Table II: List of items for subjective sensory evaluation of hair shine properties the left mastoid. All data was sampled on a laboratory computer using the Biopac AcqKnowledge software. All signals were amplified and concurrently acquired with a predefined sampling rate of 1000 Hz, supplemented by down sampling for slower biosignals (SC, ECG, and BVP). Further, a notch filter to suppress line voltage from surrounding electrical equipment and highand low-pass filtering were utilized to obtain artifact-free data. To synchronize physiological and eye-tracking recording, a hot key was programmed to start both recordings at the same time and stop them automatically after 30 s of acquisition period. After data acquisition, the physiological data were parameterized using the EDAPara software (Periphysys GmbH, Wuppertal/ Germany) for SC analysis and corresponding versions for ECG, BVP and EMG. EEG data was analyzed by means of the Vision Analyzer software to perform spectral analysis of the spontaneous EEG by means of Fast Fourier Transformation. Relevant parameters for assessment of the emotional consumer response were extracted from each biosignal (Table I). To adjust the different ranges of physiological values and account for comparability, all physiological parameters were individually z-standardized based on the individual responses to the stimuli and within the baseline assessment for each subject. This procedure further corrects the resulting values for interindividual differences in physiological responsiveness and delivers a higher degree of comparability. After each stimulus presentation, a 33-item questionnaire was filled in by the participants to assess the subjective impression of hair shine qualities by ranking the items on a 9- point Likert-type answer scale (1 = not at all; 9 = absolutely). The 33 items are compiled in Table II. Test procedure with volunteers Volunteers were recruited from an untrained consumer panel. As women are usually more involved and experienced in evaluation of hair shine and also the key target group for cosmetic suppliers, only female participants were invited. Altogether 40 female volunteers (M age = 27.8 years; SD age = 7.4 years; range: years) participated in the study. All participants were Germans of Caucasian origin. Volunteers did not receive an explicit briefing on assessing hair shine properties, but it was made clear that the investigation was about visual assessment of hair tresses. After the proper pre-experiment preparations had been done (explanation of the procedure, signing of informed consent, comfortable positioning in front of the presentation box, electrode attachment, calibration of the eye-tracking system and blocking of outside light with a black cloth), a 120-second baseline recording of the psychophysiological signals was made. Afterwards the standardized evaluation of all five hair tress stimuli began. Physiological recording and eye tracking started as soon as each sample was uncovered by the experimenter and lasted for 30 seconds. After presentation of each stimulus, participants received the 33 item questionnaire for their subjective assessment of each hair tress. This procedure was repeated for each of the five samples, with sample R always presented first as a medium-toned reference, and thereafter samples A, B, C and D in counterbalanced order. After presentation of all single samples the experiment was continued with paired presentation of two tresses, i.e. each sample A, B, C and D together with the reference sample R. During the first paired presentation run, reference sample R was presented on the left side, whereas during a second paired presentation run, reference sample R was presented on the right side. During the two paired presentation runs, eye tracking and the objective emotional assessment were recorded and followed by subjective evaluation of all samples by means of the above-described 33-item questionnaire. Statistical analysis Subjective and physiological data were subjected conjointly to discriminant analysis using SPSS The different stimulus presentations served as grouping variables, resulting in five groups to be discriminated. To account for the prerequisite of independent samples despite the repeated measurement design, matrix rotation was performed to eliminate variance owing to individual differences in response patterns [13]. Goodness of the model was evaluated by Wilk s Lambda and Eigenvalues of the extracted discriminant dimensions. The overall percentage of correct reclassifications served as an indicator of model fit, with at least twice the amount of random probability, i.e. 40%, expected for a satisfactory model. Inclusion or exclusion of variables in the discriminant model was performed with a stepwise method, assessing the contribution of each variable for the goodness of the model. The correlations of variables with the extracted discriminant dimensions were used for con- Figure 3: Upper part: Individual example of gaze patterns for the five different samples represented as heat maps. Lower part: Analysis of relative viewing time in % for each horizontal 1 mm segment of the 5.5 cm viewable area of the five samples for all subjects; color codes for viewing time on the left. IFSCC Magazine

6 Figure 4: Relative viewing time (in percent) of shine band center for the four samples A, B, C and D within the three different presentation types Figure 5: Localization of the five assessed hair tresses (group centroids) on discriminant dimensions 1 and 2 tent-related interpretation of the respective dimension. Group centroids indicated the position of the assessed tresses on each discriminant dimension, i.e. the degree of attribute specificity for the stimulus. RESULTS Eye tracking single presentation In Figure 3, an individual example of gaze pattern distribution for all five samples is displayed in a heat map representation, with red indicating areas attracting the highest attention and green for areas with medium attention. The broad variability of these patterns is obvious. Whereas the highest attention in most cases seems to be focused on the upper boundary of the shine band, thus indicating the importance of contrast as a driving force for gaze direction, especially the individual example for sample R shows that the main attention of the panelist was focused on an area on the lower part of the hair tress due to an irregularity in the tress caused by a single hair fiber that was much darker than the adjacent lighter fibers. This finding strongly suggests that regularity of a prepared tress and order phenomena play a crucial role in hair shine assessment and have to be diligently taken into account in the design of suitable experimental setups. To level out individual differences in gaze patterns and obtain statistically meaningful data, relative viewing time (in %) for each horizontal 1 mm segment of the overall 5.5 cm presented viewable area of the five samples was calculated and averaged for all subjects (Figure 3, lower part). Generally, it can be stated that shapes of gaze patterns resemble fairly Gaussian normal distributions, with the peak over the lightest area of the tress, i.e. the shine band corresponding to the maximum intensity of the light specularly reflected at the surface of the hair fibers. The highest peak, corresponding to the steepest slope of the distribution, and thus the most pronounced concentration of gazes was obtained for the blondest tress D. In contrast, the flattest peak (lowest slope of the distribution) was obtained for the reference tress R. Thus, while the gaze pattern for the reference tress R reflects a broader and detailed scanning of the whole hair tress, gazes on tress D reflect focusing on the shiniest and most attracting area of the tress. Eye tracking paired presentation Analysis of the eye tracking data obtained during the paired presentation revealed two interacting effects: Participants showed longer viewing times for stimuli located on the right side (main effect presentation side: p<.001), and reference sample R was viewed for a shorter time than the other samples (main effect reference vs. other samples: Table III: Position effects in the pairwise comparison setup; numbers given are viewing times in percent [%]. p<.001). The interaction (interaction effect presentation side x reference vs. sample: p<.001) of these two effects resulted in a gaze pattern shown in Table III. When the reference sample was presented on the left side, the viewing time in percent was longer for all the other samples. When the reference sample was presented on the right side, the viewing times for the reference and the other samples were almost equal. An interesting observation was made by visually comparing the heat map representations of the single versus the double hair presentation. In the double presentation the volunteers looked more at the center of the very bright reflection of the shine band than at the area of highest contrast especially when samples were presented on the left side (Figure 4). This effect was confirmed by a twoway ANOVA for the relative viewing time (in percent) of the shine band center. The main effect presentation (single, right side, left side) was found to be statistically significant (p=.009), whereas the main effect sample (A, B, C, D) and the interaction effect presentation x sample were not significant (p=.225, and p=.145). Discriminant analysis of psychophysiological assessment (objective emotional assessment) For evaluation of the objective emotional assessment data recorded during the single presentations of all five different hair samples, altogether twelve physiological parameters (see Table I) and ten selected subjective items from Table II (those considered most relevant for hair shine) were subjected to discriminant analysis, which revealed four significant dimensions. Dimension 1 showed an Eigenvalue of (Wilk s Lambda =.282; df = 88; p <.001) and accounted for 42.3% of the variance. Dimension 2 showed an Eigenvalue of (Wilk s Lambda =.464; df = 63; p <.001) and accounted for 27.9 % of the variance. Dimension 3 showed an Eigenvalue 6 IFSCC Magazine

7 of (Wilk s Lambda =.663; df = 40; p <.001) and accounted for 16.6 % of the variance. For dimension 4 the Eigenvalue was (Wilk s Lambda =.832; df = 19; p =.017) and accounted for 13.2 % of the variance. All pairwise comparisons between the assessed five stimuli were significant (p <.016). Correct reclassification was achieved for 60.5% of the grouped cases. As the model accounts for more correct classification than three times the random probability (20%), the model fit can be seen as satisfactory. In the following the four extracted dimensions will be described, explained, and interpreted. As an example, Figure 5 shows the location of the five assessed hair tresses (group centroids) within the discriminant space that is formed by the extracted dimensions 1 and 2. Dimension 1 The first discriminant dimension separates the reference tress R at the negative end from the other four hair tresses. Relatively high response levels of skin conductance information processing parameters, i.e. maximum SCR amplitude (-.348), sum of amplitudes (-.280) and recovery time of the maximum SCR (-.254), characterize the response pattern elicited by tress R. In terms of subjective evaluation, the negative end of the dimension is characterized by the items artificial shine (-.262) and conspicuous (-.236). The remaining four tresses are located on dimension 1 according to their color: the blonder (resp. lighter) the tress, the more it is located towards the positive end. The positive end of the dimension is characterized by activity of the facial muscles Zygomaticus major (.497) and Masseter (.427) as well as the skin conductance level (.231). While the Zygomaticus muscle indicates positive emotional valence, Masseter activation indicates induced arousal. The phasic skin conductance parameter skin conductance level indicates emotional arousal. The physiological response pattern is complemented by loadings of the subjective rating items dull (.314) and natural impression (.231). This loading pattern can be interpreted as positively arousing emotionality. In summary, dimension 1 discriminates indepth information processing from automated positive emotional response. In-depth processing was highest for sample R due to its function as the reference in the current study and rather independent of its color. With respect to the remaining four tresses, processing appeared to be the more positiveemotionally, the blonder the hair was. Further, the blonder the hair was, the less conspicuous it was rated. Dimension 2 Discriminant dimension 2 highlights the properties of the unbleached tress A located at the negative end characterized by high loadings of the items intense (-.416) and shimmering (-.398). Tress A was rated the most intense and shimmering of all five tresses, probably because of the more pronounced contrast of hair shine on the darker hair. The physiological response pattern of high heart rate variability for tress A indicates the ease of categorizing the stimulus (as dark hair) in combination with low PVA, showing that not much attention was necessary for processing. The reverse physiological response pattern was observed for tress B, located at the positive end of dimension 2. Low HRV and high PVA indicate the difficulty categorizing this one-time bleached hair, which could be described as neither dark nor blond. Tress C shows a response pattern of high HRV and high PVA indicating that, although the categorization of the sample (as blond hair) was not demanding, high attention was paid to it due to the positive emotional response it elicited during a rather automated processing mode. Dimension 3 Dimension 3 mainly separates the hair tresses C at the negative end (-.713) and B at the positive end of the dimension (.718). Tress B is characterized by high ratings of multifaceted and high Corrugator activity, indicating rather critical evaluation, whereas tress C is characterized by low Corrugator activity and high HRV. These characterizations fit well with the response patterns just described for tress B and C as being difficult versus easy to categorize. Figure 6: Physio-HeatMaps for hair tresses R and D with separated positive and negative emotional load Figure 7: Emotional Load indices from Physio-HeatMaps for all five hair samples. Index based on individually z-transformed values: negative values are below average, positive values are above average. Dimension 4 Finally, discriminant dimension 4 mainly separates the two blondest tresses, C at the positive end and D at the negative end. Both tresses trigger positively-toned emotional arousal, which is reflected in different physiological arousal indicators. Whereas responses to tress C are characterized by higher Masseter activity, responses to tress D are characterized by a high skin conductance level and low EEG Theta activity. Physio-HeatMaps As described above, discriminant analysis revealed response patterns based on integrated subjective ratings (reflecting conscious judgments based on sensory properties) as well as physiological indicators of emotional response and (cognitive) information processing. An additional analysis was conducted to highlight solely the emotional physiological responses based on valence and IFSCC Magazine

8 arousal indicators. Therefore, single indicators were standardized over the different experimental conditions (including the baseline measurement) individually for each subject by means of z-transformation ([individual response value individual mean of response values]/individual standard deviation of response values). This resulted in standardized indicators, each with a mean of zero and standard deviation of one for each individual subject over the different conditions [14] [15]. After standardization, one index was calculated for negative emotional load: [Corrugator activity + Masseter activity + heart rate + number of skin conductance responses]/4, and one index for positive emotional load: [Zygomaticus activity + heart rate variability + modulation of pulse volume amplitude + electrodermal level]/4. Results are visualized by means of Physio- HeatMaps (see examples of tresses R and D in Figure 6). Positive and negative emotional load was lowest for reference tress R. Medium emotional load was obtained for tresses A and B, with the positive and negative component being relatively balanced. Positive emotional load was highest for tress C, accompanied by low negative emotional load. Tress D (Figure 6) showed high positive emotional load accompanied by a lower negative emotional load than tress C. The results for the two emotional load indices (positive negative) for all five samples are displayed in Figure 7. From this representation, it is evident that the difference in positive and negative load indices is highest for the lightest tress D. This difference can be interpreted as an emotional index which indicates the relative strength of positive emotional load in relation to negative emotional load. DISCUSSION The current study analyzed the perception of volunteers of hair shine on Chinese hair samples with increasing degrees of bleaching, thus representing a systematic variation of reflection properties (specular reflection vs. diffuse scattering) and concomitantly hair color (increasing lightness). Several techniques were combined in an integrative approach, namely eye tracking as well as information processing and emotional response indicators extracted from psychophysiological measurements, which were additionally complemented by a subjective assessment of sensory parameters (ranking of items). In addition to discriminant analysis of combined physiological and sensory data, Physio- HeatMaps were used to highlight the physiological emotional responses based on valence and arousal indicators. This investigation shows that there is a clear separation of the shine properties evoked by the different bleaching treatments. It should nevertheless always be taken into account that there is a strong interaction between perception of hair shine with hair color, which is supported by our measurements. Furthermore, our results underline the fact that assignment of a certain stimulus within an experimental setup as reference clearly influences how it is perceived, processed and evaluated: The gaze pattern for reference tress R (bleached twice) showed a relatively broad distribution over the whole tress; it was processed in-depth and therefore rated as conspicuous and artificial and elicited weak emotional responses. In contrast, the gaze pattern for the blondest hair tress D (bleached 4x) showed focusing on/attraction to the shiniest area at the middle of the tress. It was evaluated in a highly automated emotional processing mode and elicited strong positive emotional responses. Responses were fairly comparable with patterns obtained for tress C (bleached 3x). Emotional responses to tress A (not bleached) and tress B (bleached once) were both balanced with medium intensities. While the dark tress A was rated as intense and shimmering (probably due to the strong contrast effect of the hair shine band versus the dark bulk of the tress) and easy to categorize (clearly dark hair) during information processing, tress B was difficult to categorize (neither blond nor dark) and therefore rated as multifaceted. A summary of how the different hair samples were characterized in the different modes of information processing (eye tracking), emotional load (objective emotional assessment) and sensory evaluation (ranking of items) is given in Table IV. Table IV: Summary of Characterization for All Five Hair Samples In addition to this concrete in-depth psychological characterization of the five samples investigated, a variety of fundamental effects were identified in the present study, which all presumably have significant impact on subjective hair shine evaluation in general: The hair treatment procedure of course always has a decisive influence on the subsequent characterization, leading to different degrees of hair color and lightness as well as different light reflection/scattering patterns. These different physical qualities strongly interact and most likely cannot be consciously separated by the panelists. Emotional response highly differentiates and is triggered by sensory perception, e.g. natural vs. artificial appearance. As shown by the heat map eye tracking data, the perception of hair shine is linked to certain specific areas on the presented hair tresses. Areas of highest contrast and highest reflection intensity both play dominant and interacting roles. The question whether hair shine perception is mainly triggered by contrast OR intensity thus has to be answered in a way that BOTH effects are crucial. Positioning effects in experimental setups where more than one sample is presented simultaneously strongly influence volunteers perception. As was shown in our comparative setup, the test persons perception was biased by positioning the samples on the right or left, which may be due to individual preferences or implicit assumptions on the position of the reference sample. Disturbing factors like (even only slight) irregularities of the hair arrangement (protruding hair fibers, differently colored single fibers) or artifacts like dust particles 8 IFSCC Magazine

9 in the volunteers field of view clearly attract the attention and the gaze fixation of the volunteers and therefore may have a strong influence on the evaluation of the desired experimental parameters. CONCLUSION In summary, we conclude that the combination of the two complementary techniques, namely eye tracking and recording of physiological parameters, can be used successfully to further elucidate the perception of hair shine. The results from this study may therefore be of importance for future development of suitable hair shine evaluation test protocols based on volunteers perception. As an example, the preference of test persons for a special position in a comparative presentation definitely has to be taken into account. Additionally, the influence of hair color most likely plays a major role in the evaluation of hair shine. Hence, the applied techniques look promising with regard to evaluating the performance of different cosmetic products targeting hair shine enhancement. REFERENCES: [1] Robbins, C. R., Chemical and physical behavior of human hair, Springer, New York, 2012, 5th Edition. [2] Reich, C., and Robbins, C.R., Light scattering and shine measurements of human hair: A sensitive probe of the hair surface, J. Soc. Cosmet. Chem., 44 (1993) [3] Joos, M., Rötting, M., and Velichkovsky, B.M., Die Bewegungen des menschlichen Auges: Fakten, Methoden und innovative Anwendungen, in: Rickheit, G., Hermann, T., and Deutsch, W. (Eds.), Handbuch der Psycholinguistik, De Gruyter, Berlin, 2003, pp [4] Duchowski, A. T., Eye Tracking Methodology: Theory and practice, Springer, 2007, 2nd Edition. [5] Kobayashi, N., Fukuda, R., Arai, S., Bubb, H., and Fukuda, T., What we look for in skin analysis of eye movement in skin assessment, 21st IFSCC Congress, Berlin (2000). [6] Kobayashi, N., Usui, T., Arai, S., and Fukuda, T., Analysis of eye movement in skin assessment, J. Soc. Cosmet. Chem. Japan, 36 (2002) [7] Mika, N., Rie, H., and Ryoko, F., Proposing a new eye-catch makeup concept human impressions alter by visual guidance using makeup, 26th IFSCC Congress, Buenos Aires (2010). [8] Misaki, E., Ikeda, H., and Imai, T., Analysis of the actual status of make-up with an eyetracking system study on eye-tracking analysis of beauty experts, J. Soc. Cosmet. Chem. Japan, 46 (2012) [9] Hagens, R., Wiersbinski, T., Becker, M. E., Weisshaar, J., Schreiner, V., and Wenck, H., Qualification of an automated device to objectively assess the effect of hair care products on hair shine, J. Cosmet. Sci., 62 (2011) [10] Fink, B., Neuser, F., Deloux, G., Röder, S., and Matts, P. J., Visual attention to and perception of undamaged and damaged versions of natural and colored female hair, J. Cosmet. Dermatol., 12 (2013) [11] Eisfeld, W., Wachter, R., Schaefer, F., and Boucsein, W., Objective Emotional Assessment of Perceivable Wellness Effects, Cosmetics & Toiletries Magazine, 122 (2007) [12] Boucsein, W., Schaefer, F., Schwerdtfeger, A., Busch, P., and Eisfeld, W., Objective Emotional Assessment of Foam, SÖFW- Journal, 125 (1999) [13] Boucsein, W., Schaefer, F., Kefel, M., Busch, P., and Eisfeld, W., Objective emotional assessment of tactile hair properties and their modulation by different product worlds, Int. J. Cosmet. Sci., 24 (2002) [14] Schmidt, J., and Opwis, M., Insight inside - Was der Körper verrät. Alternativen zur bildgebenden Hirnforschung, planung & analyse, 6 (2011), [15] Stürmer, R., and Schmidt, J., Erfolgreiches Marketing durch Emotionsforschung, Haufe, München, Corresponding author IFSCC Magazine

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11 Decorative Cosmetics: In Vivo Facial Measurement of Color Parameters, Even Skin Tone and Radiance Stephan Bielfeldt, Marianne Brandt, Nathalie Lunau, Matthias Seise, Gunja Springmann, Klaus-P. Wilhelm proderm Institute for Applied Dermatological Research, Schenefeld, Germany Parts of this publication were presented as a poster presentation at the 28. IFSCC Congress, October 27-30, 2014, Paris, France. Keywords: Color fading, radiance, even skin tone, image analysis, trained raters, lay raters ABSTRACT Color cosmetics like mascara, make-up and lipstick are promoted with claims like "long-lasting", "high covering power" or "improve attractiveness". Makeup, foundations and powder products further are claimed to improve the evenness of the skin or reduce the visibility of wrinkles. Especially subjective parameters like radiance of facial skin have gained importance. In this article we describe a portfolio of in vivo methods to support such claims for decorative cosmetics. INTRODUCTION The outward appearance of the body and especially the female face has increasingly gained the interest of research [1-6]. Its importance for personal well-being, social status and reputation is nowadays well understood. Since prehistoric times, it has been believed that cosmetic treatments can improve the outward appearance, and it is not surprising that for about 40 years the role of cosmetics has been investigated intensively in this context [1-25]. In the last decades the cosmetic industry has continuously widened the range of attributes and beauty claims for decorative cosmetics. Classical color cosmetics like mascara, makeup and lipsticks are promoted with claims like "long-lasting", "high covering power" or "improve attractiveness". Makeup foundations, tinted day creams and powder products additionally are claimed to improve the evenness of the skin or to reduce the visibility of wrinkles and pores [31]. A further important beauty parameter of facial skin is its radiance [26]. As a subjective perception, it includes not only physical and physiological aspects like complicated lightskin interactions but also psychological aspects of the observer. Therefore radiance is difficult to define and quantify [27]. It is generally assumed that beautiful skin radiance is acquired by the combination of a skin The assessments are based on high performance facial images. The quality of the photographs enables direct image analysis measurements of color parameters on unbiased digital images and a highly discriminative standardized rating of subjective parameters. Image analysis of photographs transferred to the CIE L*a*b* color space are suitable to quantify the permanence of decorative cosmetics on facial skin. The fading of lipstick, mascara and makeup/foundation products could be precisely quantified by newly developed image analysis tools. The results compared well with lay person ratings of surface component of light reflection that makes the skin look shiny and a second specific subsurface component that is seen in fair skin [26]. All beauty parameters like skin radiance are influenced by inhomogeneous attributes of skin like blemishes, pigmented spots or wrinkles [28]. A more indirect and more broadly distributed backscatter of light from skin due to a cosmetic treatment, the so -called soft focus effect [29], evens out inhomogeneous aspects of skin and leads to a more beautiful shine and radiance. High scores for radiance are given for facial areas that are light but not white. There must still be a good visibility of the color of the face [30]. Another beauty parameter that has gained increased importance is attractiveness. The evaluation of an improvement of attractiveness with use of cosmetics is one of the core research fields of the cosmetic industry [1, 3-6, 8, 10-13, 16, 25]. The use of cosmetics influences the consideration of attractiveness in the eye of the beholder as well as user behavior [8, 23]. Women wearing makeup are considered to be more confident and wealthier and have a higher job position than peers without makeup [23]. Mainly decorative cosmetics increase facial attractiveness of women by increasing facial contrast [1, 2, 10]. Cosmetics like mascara and lipstick further enhance this contrast [1]. A third parameter is evenness. The more homogeneous the texture of the skin appears, the more beautiful it is rated [5, 6, 13]. the same images in most assessments. The lay and expert rating of the photographs for the parameters attractiveness and skin radiance revealed a high discrimination power between treatments and no relevant differences between the results of experts and lay persons. It can be concluded that the reported image analysis tools and rating methods are well suitable to assess the investigated objective and subjective parameters for decorative cosmetics. Especially the lay rating methods enable results close to the consumer perception. In this article we describe a portfolio of in vivo methods to measure the efficacy of decorative cosmetics. We have chosen parameters and tools which are sensitive and reliable and can be well understood by the consumer. Our assessments are based on high performance facial images. The quality and reproducibility of the obtained images are of paramount importance to enable direct image analysis measurement of color parameters on the unbiased digital images. Standardized rating of subjective parameters by trained graders and lay persons is an alternative approach for evaluating various parameters in a highly discriminative and reproducible way. EXPERIMENTAL Facial photography A custom-built unit for standardized and reproducible clinical photography (USR-CliP) was used for standardized photography. The device enables high reproducibility of subject repositioning due to a fixed support and a ghost image tool that can display facial overlay images. Upon repetition of a photograph, i.e. after treatment, a baseline image can be used as an overlay of the online image to adjust the subject exactly to the baseline position. The main components of the photographic system are a Hasselblad camera H4D31 with 31 megapixel (4,872x6,496) IFSCC Magazine

12 physical resolution and 16 bit color resolution of the Charge-coupled Device (CCD) chip (Hasselblad, Ahrensburg, Germany) in combination with a professional flash system (Broncolor Scoro A2, Broncolor Picolight and Broncolor ringflash C) which allows flashes of a constant color temperature (5500 Kelvin) independent of flash time and light intensity. Images were captured in raw format, and a small color standard plate (X-Rite ColorChecker passport) was photographed as a part of each image. For the color consistency check, white balancing and color calibration, separate photographs of the X-Rite ColorChecker Classic were used. Image rating of experts and lay persons on color calibrated screens Image rating was performed in a standardized setup on color calibrated screens (NEC SpectraView Reference 3090, 30", 2,560 x 1,600 pixel, 99.6% Adobe RGB). The images were cropped to the visible face and downsampled to 1,600 pixel height to match the screen resolution. The graders were placed at a fixed distance of 50 cm in front of the screens in a quiet room with dimmed light. Assessment of color homogeneity and color fade by image analysis and rating of photographs To evaluate the time-dependent color fade of cosmetic products, different types of makeup products, low price and high price makeup and lipstick were purchased on the market and applied by a beautician to the faces of 12 female subjects (aged 19 to 39 years). Since no other criteria for product preselection were available, it was assumed that the price is a suitable indicator to find test products with different performance in color stay. Figure 1 illustrates the test design. Before application, after 10 minutes as well as 1, 3, and 6 hours after application clinical facial photographs of the subjects were taken as described above. The images were rated by 6 trained experts and by the 12 subjects themselves (n=18) on defined parameters to assess the hold of the products over the investigated period of time. Color image analysis was performed after transforming the color of each pixel to CIE L*a*b*. The covering power of lipstick and lip gloss were assessed using the redness parameter a* on the interactively marked lip region. The yellowness b* was used to assess the homogeneity change of the makeup products for assessment of the covering power of makeup by image analysis. The measurement areas were selected by interactive marking of the forehead and cheeks (excluding the eyes, nose and mouth). The variation of the color (1/standard deviation) was assessed as the parameter for homogeneity using a moving window approach. In detail, the feature was calculated for each pixel as the inverse of the standard deviation of all neighboring pixels. Rating of radiance, attractiveness and even skin tone on facial photographs To investigate the appropriateness of the photographic unit and the standardized rating methodology of trained and lay raters, six facial leave-on cosmetics were purchased from the market. Only products that contained ingredients declared to increase facial radiance were chosen. Because there was no previous knowledge on the performance of these products, in the first step the goal was to find a candidate that showed improvement of facial radiance in a pre-study. A stepwise procedure with expert rating of pairs of Figure 1: Photographs of a representative subject illustrating the test design. Reproducible high quality photographs were taken before and 10 minutes, 1 hour, 3 hours and 6 hours after application of the test products. On two days different types of makeup were applied. Images in the top row show the low price set (low-price mascara, tinted day cream, lip gloss). In the bottom row the high price set (high-price mascara, cream makeup, lipstick) is presented. facial photographs was performed to preselect the candidates. In the first step one stimulus person per test product was photographed before and after application of the respective test product. The applications were performed by a beautician in accordance with the instructions for use of the suppliers. Three expert raters selected the product with the best result in enhancement of radiance and even skin tone. Based on this rating (data not shown), two candidates remained that could not be discriminated. In a second run, the experiment was repeated on 6 stimulus persons and only the two remaining products were tested. The three experts evaluated the best performing product (data not shown). This product was selected for the main study. The pre-selected product was evaluated for facial radiance, attractiveness, and evenness of the skin tone on standardized facial photographs by a panel of 27 female stimulus persons (aged 26 to 49 years, mean age: 40.4 years, SD: 6.6 years). Lipstick users were allowed to apply lipstick to avoid artificial standardization in the study and to investigate the influence of lipstick use on the results. In total, 11 of the subjects applied no lipstick, while a group of 16 subjects was photographed after freshly applied lipstick. One of four different nuances of the test product was chosen for each subject according to a good fit of the lightness of color to the skin type. In all cases, the nuance was chosen that was slightly lighter than the natural skin tone. After taking baseline photos, the test product was applied to the face of the stimulus persons by a beautician. Photographs were taken again after a two-hour waiting time to make sure that the products had soaked in and dried completely. A group of lay raters (N=24) and a group of trained graders (N=6) assessed skin radiance, attractiveness, and evenness of facial skin tone. Photographs of the 27 female subjects were presented blinded and randomized in pairs (untreated versus treated) on color-calibrated monitors. Both rater groups ranked each pair of images with either "1" (prefer) or "0" (not prefer) separately in three runs for the parameters radiance, attractiveness and even skin tone. Statistics Mean values and standard deviations as well as 95% confidence limits were calculated from the raters' evaluations of radiance, attractiveness and evenness using the statistics software SAS 9.2 (SAS Institute, Cary NC, USA). A subgroup analysis was performed to analyze a possible interfering effect of enhanced lip color due to lipstick application. 12 IFSCC Magazine

13 tween both products directly after application compared with untreated skin as a control (Figure 4 B). The makeup showed a higher covering power than the tinted day cream. However the rate of decrease of covering power was comparable for the low-priced tinted day cream and the high-priced makeup product as observed in both rating and image color analysis (Figure 4 B). Figure 2: Assessment of covering power of lipstick (A) and makeup (B) by raters. The image was shown together with control images for 0% and 100%. No covering power (0%) is seen when no products are applied. The highest covering power is seen 10 minutes after application (100%). The rater selects the appropriate value electronically on a slider. A rating bias was observed in the subjective ratings (Figures 3 and 4). The first decrease in the subjective assessment is presumably attributable to the procedure for presenting the images to the raters because this procedure implicated the assumption that there should have been a difference between the images. With this implication in mind, the graders never rated any parameter with 100%. Figure 3: Comparison of subjective (A) and objective (B) assessment of lasting of lipstick/lip gloss. RESULTS Assessment of color homogeneity and color fade by image analysis and rating of photographs The results showed that both low-priced lip gloss and higher priced lipstick faded in color intensity during the six-hour assessment time after application (Figure 2 A). Based on the image analysis, the lip gloss had a lower covering power than the lipstick (Figure 3 B). The main color fade occurred during the first three hours, while during the following three hours color decreased further only slightly (Figure 3). Over the complete assessment time a clear difference between the covering power of lip gloss and lipstick was seen with a clearly better hold of the lipstick product. The rating data correlated with the data of the image color analysis (Figure 3 A and B). Correspondingly, the rating data relating to makeup and tinted day cream showed the main decrease in covering power in the first three hours after the product application. During the following three hours only a slight decrease in the covering effect was seen (Figure 4). The results of the image color analysis were superior to the visual rating results (Figure 4 B). It was possible to show clear differences in the covering power be- Judgment of Radiance, Attractiveness, and Evenness of Skin Tone on Facial Photographs by Lay Raters and Trained Graders The study was performed on one preselected test product as described above. Figure 5 shows as representative examples. Pairs of photographs of two stimulus persons, untreated and treated with the test product, as representative examples of the obtained results. Table I gives an overview on the descriptive statistics of the test results. Figure 4: Comparison of subjective (A) and objective (B) assessment of covering power of makeup. Figure 5: Reproducible high-quality photography before and after application of the test product. Photographs of two representative subjects without (upper image, left side) and with test product (upper image, right side) were presented to the raters on color calibrated monitors as a pair in a blinded and randomized manner. Raters subsequently judged which of the two images they preferred in terms of radiance, attractiveness and even skin tone. The lower pair of images shows a subject using lipstick (test product applied is shown in the right image). IFSCC Magazine

14 Table I: Rater judgment (in % of raters choosing the image with treated face to be superior) for radiance, attractiveness, and evenness of skin tone; trained graders N=6; Lay: lay raters N=24; N: number of stimulus persons; SD: standard deviation; CI: confidence interval better attractiveness where subjects wore the test product. Rating of the images where stimulus persons used lipstick was almost comparably (Figure 7 A). The respective results with lipstick were 88% and 95% (Figure 7 B). Discrimination of attractiveness was slightly better than of the parameter radiance. The third beauty parameter judged was even skin tone and it is presented in Figure 8. Both the lay raters (89 %) and trained raters (91 %) subjectively rated images with a more even skin tone where subjects wore the test product. Rating of images where stimulus persons used lipstick was almost comparably (Figure 8 A). The respective results with lipstick were 88% for both rater panels (Figure 8 B). Discrimination of even skin tone was similar to that of the parameter radiance. Figures 6 to 8 show the results of Table I in graphs displaying the means and confidence limits. The first beauty parameter judged was skin radiance and it is presented in Figure 6. Both the lay raters (81 %) and trained graders (92 %) subjectively rated images with better radiance where subjects had on the test product. Rating of the images where stimulus persons used lipstick was almost comparable (Figure 6 A). The respective results with lipstick were 77% and 89%. (Figure 6 B). The second beauty parameter judged was skin attractiveness and it is presented in Figure 7. Both the lay raters (91 %) and trained graders (98 %) subjectively rated images with DISCUSSION AND CONCLUSIONS One intention of this study was to explore the suitability of lay person and expert ratings of photographs to assess the fading of color cosmetics and to compare the results with objective image analysis measurements. Regarding the assessment and measurement of color fade, we conclude that the hold and longlasting claim of decorative cosmetics can be determined in a reproducible way by subjective rating and objective image analysis of high-quality clinical photographs when they are presented in a blinded way and as a direct comparison of times or treatments. For assessing the fading of makeup products over the wear time, image analysis of color homogeneity is a much better method than visual image rating. Figure 6: Percentage of raters seeing better radiance with the test product applied (dark blue) or untreated (light blue); (A) Stimulus persons without lipstick (N = 11): (B) Stimulus persons with lipstick (N = 16). Figure 7: Percentage of raters seeing better attractiveness with the test product applied (dark blue) or untreated (light blue); (A) Stimulus persons without lipstick (N = 11): (B) Stimulus persons with lipstick (N = 16). Our second intention was to find a way to easily identify cosmetic products that improve facial radiance. Based on a published multicenter study on attractiveness evaluation (28) we used paired image ranking to explore all three subjective parameters. The results of our assessments confirm the power of this methodology. Our selection of product candidates based on facial images was successful, even though only 4 stimulus persons were photographed. The larger panel of lay and trained raters confirmed that the selected product provided radiance. Further studies might reveal the robustness and reliability of our screening evaluation method to successfully select candidate products for the beauty claims investigated here. We believe that a low number of stimulus persons and expert raters are already sufficient for such a pre-selection process. 14 IFSCC Magazine

15 Figure 8: Percentage of raters seeing more even skin tone with the test product applied (dark blue) or untreated (light blue); (A) Stimulus persons without lipstick (N = 11): (B) Stimulus persons with lipstick (N = 16). Our third intention was to find parameters that are related to radiance. We estimated that attractiveness is a wider concept and radiance one source of attractiveness. We further assumed that a positively judged increase in radiance can not only come from a lightening of the skin tone but would also require a more even skin tone. Although our results do not generate causality, we were able to demonstrate that the investigated product that increased skin radiance also produced a increase in attractiveness and evenness of skin tone. Image rating is a subjective method and relies on the qualities of the raters, but as can be seen from the very homogeneous ratings by trained and lay raters, even the human eye of a lay person is able to very robustly assess the investigated beauty parameters from highly standardized pairs of photographs. This is not even biased when stimulus persons use other cosmetics as lipsticks. While attractiveness assessments seem to be quite unaffected by ethnic and cultural influences (32), this is not clear for the parameter radiance. In future studies on larger rater panels of different ethnic origins and comparisons of evaluations in different countries might clear up this point. 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16 [27] Baret, M., Bensimon, N., Coronel, S., Ventura, S., Nicolas-Garcia, S., Korichi, R., and Gazano, G., Characterization and quantification of the skin radiance through new digital image analysis, Skin Res. Technol., 12 (2006) [28] Bielfeldt, S., Henss, R., Koop, U., Degwert, J., Heinrich, U., Jassoy, C., Meyer, J., Tronnier, H., Jentzsch, A., and Blume, G., Internet-based lay person rating of facial photographs to assess effects of a cleansing product and a decent cosmetic foundation on the attractiveness of female faces, Int J Cosmet Sci, 35 (2013) [29] Becker, M., Schmidt, C., Hochstein, V., and Petsitis, X., A novel method to measure and pre-select functional filler pigments, Cosmetics & Toiletries magazine, 127 (2012) [30] Ikeda, N., Miyashita, K., Hikima, R., and Tominaga, S., Reflection measurement and visual evaluation of the luminosity of skin coated with powder foundation, Color Res. Appl, 39 (1) (2014) [31] Draelos, Z.D., The cosmeceutical realm, Clin Dermatol, 26 (6) (2008) [32] Thornhill, R., and Gangestad, S.W., Facial attractiveness, Trends in Cognitive Sciences, (1999) Corresponding author 16 IFSCC Magazine

17 Swertia chirata Extract Promotes Epidermal Regeneration through Stimulation of Paracrine Communication between Adipose-Derived Stem Cells and the Epidermis Carine Bézivin 1, Arléty Pérolat 1, Estelle Loing 2 1 Lucas Meyer Cosmetics, ZA les Belles Fontaines, 99 route de Versailles, Champlan, FRANCE 2 Lucas Meyer Cosmetics, Tour de la Cité, 2600, boulevard Laurier, suite 900, Québec, G1V 4W2, CANADA Keywords: Hypodermis, stem cells, paracrine communication, KGF/FGF-7, anti-aging strategy ABSTRACT During the last years, adipose-derived stem cells from the subcutaneous adipose tissue have been identified and characterized as key players in the stimulation of re-epithelialization during wound healing, enhancing keratinocyte proliferation and migration through a paracrine communication mechanism. KGF/FGF-7 INTRODUCTION Growth factors and cytokines are vital mediators of cellular communication. In the skin they are involved in wound healing, continuous regeneration and repair processes. Chronological skin aging is characterized by a decrease in these growth factors, leading to a thinner epidermis which accelerates wrinkle appearance [1]. Many cells are able to synthesize them and numerous factors have been widely studied, including in keratinocytes, fibroblasts, endothelial cells, macrophages and platelets. Recently, studies on reconstructive surgery have highlighted new contributors in this process, i.e. adiposederived stem cells from the subcutaneous adipose tissue, a population of pluripotent mesenchymal cells [2]. Adipose-derived stem cells are able to stimulate re-epithelialization during wound healing by enhancing keratinocyte proliferation through cell cycle progression and migration [3]. This effect is mediated at least partially by paracrine action of KGF/ FGF-7 [4, 5, 6]. A previous transcriptome-wide study on human adipocytes led to the identification of a natural molecule from the iridoid family, swertiamarin, which is purified from Indian gentian, Swertia chirata (Gentianaceae). Gene expression data demonstrated the capacity of a Swertia chirata extract (SWT) titrated in swertiamarin ( 90%) to induce multiple genes known to be involved in the inhibition of the differentiation of uncommitted mesenchymal stem cells, i.e. members of the histone deacetylase complex, histone demethylases, the telomere-associated protein RIF1, the stem cell specific ETV5 transcription factor, the Kruppel-like factor 4 (KLF4) and many others [7, 8, 9, 10, 11, 12]. The data constituted a bundle of evidence suggesting that Swertia chirata extract might be a powerful stemness maintenance factor, thereby limiting the rate of adipocyte differentiation. In addition, the data demonstrated a sustained increase in KGF/FGF-7 mrna, coding for keratinocyte growth factor, and also in FGFR1 mrna, its receptor. Together they promote preadipocyte proliferation via an autocrine mechanism [13]. In the present paper, we provide in vitro evidence that Swertia chirata extract promotes soluble KGF/FGF-7 release from subcutaneous adipocytes and favors epidermal reepithelialization through a paracrine communication pathway. When formulated and topically applied to skin explants, Swertia chirata extract stimulates a retinoic-like activity, with an increase in the number of cell layers and a slight densification of the papillary dermis. This stimulation is clearly enhanced when Swertia chirata extract is applied to non-dermatomized skin containing the adipose compartment and adiposederived stem cells, suggesting that the compound also directly stimulates the adiposederived stem cells, which in turn activate the epidermis. In addition, studies on human volunteers demonstrated that, compared with placebo, a 0.025% Swertia chirata extract cream shaded the appearance of vertical wrinkles and improved both skin texture and wrinkle volume. The paper highlights an derived from adipose-derived stem cells plays a major role in this process. In this study, we provide in vitro evidence that Swertia chirata extract promotes release of soluble KGF/FGF-7 from subcutaneous adipocytes and favors epidermal re-epithelialization. We also demonstrate ex vivo that Swertia chirata extract increases epidermal thickness in an adipose tissue-dependent mechanism. In clinical trials Swertia chirata extract reduced the appearance of vertical wrinkles on the lower face area and lip contour with a concomitant reduction of wrinkle volume and improved texture after only seven days. original mechanism of action to promote epidermal stimulation and renewal, through a cross-talk between the adipose tissue and the epidermis based on growth factor secretion and paracrine communication. EXPERIMENTAL Cell culture and treatment Normal human adipocytes were isolated from an abdominoplasty from a 34-year-old woman aged and cultured in Minimum Essential Medium (MEM, Sigma). Swertia chirata extract was dissolved at 10% (w/v) in DMSO and applied within the adipocyte culture medium at , and % (w/v) for 15 hours, with a constant concentration of 0.15% DMSO (v/v). DMSO 0.15% was used as the control condition. Depending on the experiment, the medium was then collected for further use. Normal human epidermal keratinocytes (NHEK) were isolated from an abdominoplasty performed on a 45-year old woman, grown in Keratinocyte Growth Medium 2 (KGM2, PromoCELL), and maintained in a humidified incubator at 37 C with a 5% CO 2 atmosphere. In vitro re-epithelialization assay At cell confluence, a scratch was made in each keratinocyte culture well by scraping, allowing the formation of a cell-free area. The cultures were then incubated at 37 C with the Swertia chirata extract-treated adipocyte culture medium diluted 1:10 in the keratinocyte medium, without prior inhibi- IFSCC Magazine

18 KGF/FGF-7 (pg/ml) tion of cellular proliferation. After 72 hours of incubation the effect was evaluated by comparison with an untreated control (containing 0.15% DMSO). Pictures were taken at the beginning of the experiment and at the end of the contact period (72). The Image J software was then used for quantification of the colonized areas after treatment. The statistical significance of differences between the DMSO and Swertia chirata extract specimens was assessed by analysis of variance (One Way ANOVA) followed by a Holm Sidak test (* p<0.05). ELISA KGF release was quantified using a sensitive and specific ELISA kit according to the manufacturer s instructions (RayBiotech, ref. ELH- FGF-7-001). The statistical significance of differences between the conditions DMSO and Swertia chirata extract was assessed by analysis of variance (One Way ANOVA) followed by a Holm Sidak test (* p<0.05). Ex vivo skin preparation and treatments Full skin pieces (with the epidermal, dermal and hypodermal compartments) and skin pieces (dermis and epidermis) of an average diameter of 10 mm were prepared from an abdominoplasty from a 47-year-old Caucasian woman with a phototype II/III (reference: P1144-AB47). The explants were maintained in BEM medium (BIO-EC s Explants Medium) at 37 C in a 5% CO 2 atmosphere. The test products, a cream containing 0.025% Swertia chirata extract and a placebo (the same formula without Swertia chirata extract), were applied topically for 9 days. They were re-applied twice daily (1 μl) after 1, 2, 3, 6 and 7 days. All experiments were performed on triplicate skin discs. Samples from the culture medium were collected after 3, 6 and 9 days of contact and pooled for further KGF/FGF-7 measurements. Skin biopsies were collected at day 0 and at day 9 and kept in formaldehyde 4% Histological processing DMSO SWT % SWT 0.005% SWT0.0075% Figure 1: KGF/FGF-7 measurement by ELISA from the culture medium from normal adipocyte upon treatment with 3 concentrations of Swertia chirata extract (SWT) for 15 h. The results represent the mean of 3 independent replicates and the error bars represent the standard deviation around the mean. The statistical analysis is an ANOVA followed by a Holm Sidak test. Data with a p value <0.05 were considered statistically significant (*). Tissue samples were dehydrated and impregnated in paraffin using an automatic Leica TP 1010 dehydrator. They were then embedded using a Leica EG 1160 embedding station. 5 μm-thick sections were prepared using a Leica RM 2125 Minot-type microtome and the sections were then mounted on Superfrost Plus silanized glass slides (Thermo Scientific, ref ). The general tissue morphology was examined using a Leica DMLB microscope after a Masson s trichrome staining. In vivo studies on lower face vertical wrinkles and skin texture The first study was carried out with a panel of 17 healthy Caucasian female volunteers aged between 45 and 60 years to evaluate the anti -wrinkle properties of the Swertia chirata extract cream on both half-lower faces. In a randomized, split-face study a 0.025% Swertia chirata extract cream and placebo (Table I) were applied twice a day over a period of 28 days (morning and evening). On days 7, 14 and 28, the volume severity of vertical wrinkles was measured using a three-dimensional fringe projection method. Statistical analysis consisted in comparison of the values obtained on the zones treated with each product at the different time points with the values before application. Data were analyzed with a paired student t-test. In addition, the same protocol was used on a panel of 17 healthy Caucasians aged years to assess the global appearance of half lower faces. This study was performed on days 7 and 28 using the VISIA-CR facial imaging system (Canfield) with a specific filter application for skin texture and visible appearance of wrinkles. In vivo evaluation of the anti-wrinkle effect of Swertia chirata extract on the lip contour A randomized, split-face study was performed with a group of 10 selected healthy Caucasian female volunteers aged years who smoked and had deep wrinkles around the lips. The 0.025% Swertia chirata extract cream and the placebo were applied twice a day as above for 28 days. On days 0, 7 and 28, a hemi-face analysis of the skin relief variation was performed using the Primos 3D Pico imaging system (GFMesstechnik). * Table 1: Composition of the Swertia chirata extract cream and the placebo used for clinical evaluation RESULTS AND DISCUSSION Swertia chirata extract promotes soluble KGF/FGF-7 release from subcutaneous adipocytes KGF/FGF-7 release was quantified by ELISA from the culture medium from normal adipocytes after treatment with three concentrations of Swertia chirata extract for 15 h (Figure 1). The data demonstrated a statisti- 18 IFSCC Magazine

19 % of re-epithelialization vs. T DMSO CM SWT % CM SWT % CM SWT0.0075% CM Figure 2: Re-epithelialization of a cell-free area by NHEKs keratinocytes after a 72-hour contact period with Swertia chirata extract (SWT)conditioned media (CM) collected from normal adipocytes. Conditioned media were diluted 1/10 in the keratinocyte culture medium. Left: Pictures taken at T0 and T72h; Rright: Quantification of the cell-free area by image analysis. The results represent the mean of 3 independent replicates and the error bars represent the standard deviation around the mean. The statistical analysis is an ANOVA followed by a Holm Sidak test. Data with a p value <0.05 were considered statistically significant (*). cally significant increase in KGF/FGF-7 release of 16.6% upon stimulation with % of Swertia chirata extract (* p<0.05) compared with the control condition containing 0.15% DMSO. Swertia chirata extract-treated adipocyte conditioned media (CM) promotes in vitro epidermal re-epithelialization Re-epithelialization can be studied in vitro using confluent cultures of epidermal keratinocytes. At the beginning of the test a cell-free surface is generated by scraping the cells, simulating a wound. Then the ability of the cells to close the acellular surface is measured by picture analysis, either during the re-epithelialization process or at the end of the incubation period. od the tissues were embedded in paraffin and sections were analyzed in order to determine their morphology. On day 0 and after 9 days in culture the untreated control tissues presented a similar morphology, with 3/4 cellular layers and a clear relief of the dermal -epidermal junction. The papillary dermis showed thin collagen bundles forming a moderately dense network. The dermal cells presented a good morphology (Figures 3a). The placebo cream did not induce significant changes in the morphology (Figures 3b). Remarkably, topical Swertia chirata extract induced hyperplasic acanthosis, i.e. an increase in the number of cell layers, resulting in an increase in the epidermal thickness. This epidermal stimulation, closely related to a retinoic-like activity, was clearly enhanced when adipose tissue was present, with an increase in the number of cell layers of 10/12 compared with the 6/7 layers observed when the adipose compartment was removed (Figures 3c). This data demonstrated that adipose tissue and adipose-derived stem cells play a major role in optimization of epidermal stimulation by Swertia chirata extract, with initial activation of adipocytes, which in turn activate the epidermal cells, probably through paracrine stimulation. In order to strengthen this hypothesis, tissue culture media were collected on day 9 from the different skin discs (single culture per condition), treated or not with Swertia chirata extract, for KGF/FGF-7 ELISA dosage. Low levels of KGF/FGF-7 were detected in the culture media from the explants without Swertia chirata extract-conditioned media were collected from normal adipocytes, diluted 1/10 and applied to the wounded keratinocyte cultures without any other growth factor or culture supplement. The cell -free area was measured before application, and after a 72 h contact period. The data revealed a statistically significant increase in the colonized area after 72 h of contact with the conditioned media (Figure 2). This was observed for the media conditioned with Swertia chirata extracts at % (+65%), 0.005% (+110%) and % (+55%). Topical Swertia chirata extract increases epidermal thickness ex vivo through stimulation of adipose tissue Swertia chirata extract formulated at 0.025% in a cream was applied topically for 9 days to ex vivo skin explants containing or not containing the adipose tissue compartment. At the end of the stratum corneum contact peri- Figure 3: Epidermal morphology of ex vivo skin after topical application of a 0.025%-Swertia chirata extract (SWT) cream vs. placebo IFSCC Magazine

20 Variations (% vs. placebo) Variations (% vs. placebo) Variations (% vs. placebo) KGF/FGF-7 (pg/ml) Placebo cream 0.025% SWT cream Swertia chirata extract on wrinkles. Repeated application of the 0.025% Swertia chirata extract cream for 28 days blurred the appearance of vertical wrinkles from the lower face area. Indeed, the profilometry study showed a significant reduction of the volume of vertical wrinkles after 28 days (-12% compared with placebo, * p<0.1). This study showed that the effect was progressive, as significant results were already detected after 14 days (- 10% compared with placebo, * p<0.1) (Figure 5A). Furthermore, image capture with specific filters from VISIA-CR showed that application of the 0.025% Swertia chirata extract cream significantly reduced the visible appearance of vertical wrinkles (-16% compared with placebo, ** p<0.05) (Figure 5B). Swertia chirata extract also improved skin texture, with a significant decrease in skin roughness observed after 28 days (-9% compared with placebo, ** p<0.05) (Figure 5C). hypodermis, with no major difference between tissues treated or not treated with Swertia chirata extract determined. Remarkably, there was a strong increase KGF/FGF-7 release when the adipose tissue and Swertia chirata extract were present, with a 2.7-fold stimulation upon treatment with Swertia chirata extract observed (Figure 4). Clinical studies 50 0 Ex vivo skin E+D Two different studies highlighted the effect of Ex vivo full skin E+D+H Figure 4: KGF/FGF-7 release from ex vivo skin treated for 9 days with a 0.025%-Swertia chirata extract (SWT) cream vs. placebo (E+D stands for dermis + epidermis; H stands for hypodermis). 0% The study with a focus on the lip contour based on 10 smoking female volunteers who were smokers with deep wrinkles in the area also demonstrated a significant reduction in skin roughness after 28 days (-19%, ** p<0.05) (Figure 6 left) and in wrinkle depth both after 7 (-14%, * p<0.1) and 28 days (- 15%, * p<0.1) (figure 6 right) compared with placebo. CONCLUSION Skin is a complex organ, tightly connected to multiple systems, body organs and tissues, such as the innate and adaptive immune system, the microbiota, the microvascular network or the central nervous system. Exchange of information or molecular signals between the different cell types takes place via a very complex network of small mole- -2% -4% -6% -8% -10% -12% -14% -6% -10%* -12%* Day 7 Day 14 Day 28 Figure 5: Clinical studies on the lower face area. (A) Evaluation of the vertical wrinkle volume (print analysis by fringe projection) 0% -1% -2% -3% -4% -5% -6% -7% -8% -9% -10% -5% -9%** (C) Evaluation of skin texture improvement = roughness reduction (picture analysis with VISIA CR filters). Day 7 Day 28 0% -2% -4% -6% -8% -10% -12% -14% -16% -18% -10%* -16%** (B) Appearance of vertical wrinkles from the lower face area (picture analysis with VISIA CR filters)cream and with placebo Day 7 Day 28 The statistical analysis consisted in comparison of the values obtained on zones treated with Swertia chirata extract (SWT) cream and with placebo compared with the values before application. Data were analyzed using a paired student t-test with * p<0.1 and ** p< IFSCC Magazine

21 Variations (% vs. placebo) Variations (% vs. placebo) 0% -13% -2% -4% -6% -8% -10% -12% -14% -11% Day 7 Day 28-14% -14% -14% -14% -14% -15% -14% * Day 7 Day 28-16% -15% -18% -20% -19%** -15% -15% -15% * Figure 6: Clinical study to evaluate the anti-wrinkle effect on the lip contour. Left: Analysis of skin roughness (average roughness) Right: Analysis of vertical wrinkles (average relief). Data were analyzed using a paired student t-test with ** p< 0.05 and * p< 0.1 cules or soluble proteins, such as neurotransmitters, neuropeptides, cytokines, chemokines and growth factors. In particular, there is a major connection between both the dermis and the epidermis and the adipose tissue. A tightly regulated stimulation of skin cells is ensured especially during the wound healing process by soluble factors secreted by adipose-derived stem cells, with a major contribution to reepithelialization and epidermal renewal coming from KGF/FGF-7. The action of KGF/FGF-7 on keratinocytes is mediated by a paracrine activation mechanism. In the present study we highlight the ability of the swertiamarin-rich Swertia chirata extract (Swertia chirata extract) to stimulate cross-talk between adipose-derived stem cells and epidermal keratinocytes. In vitro and ex vivo data demonstrate that Swertia chirata extract is able to activate adiposederived stem cells to promote the release of KGF/FGF-7, improve epidermal thickness and enhance the re-epithelialization phase that occurs during wound healing. Clinical tests demonstrate the ability of Swertia chirata extract to reduce the appearance of vertical wrinkles and improve skin texture with a concomitant reduction of wrinkle volume after only seven days. Fringe projection analysis also highlights a significant reduction of wrinkles on the lip contour and skin roughness. In summary, our data form a strong bundle of evidence that the addition of Swertia chirata extract to cosmetic formulations constitutes an efficient anti-aging strategy based on stimulation of paracrine communication with a never-seen-before mechanism of action. REFERENCES [1] Stanulis-Praeger, B.M., and Gilchrest, B.A., Growth factor responsiveness declines during adulthood for human skin-derived cells, Mech. Ageing Dev, 35 (1986) [2] Zhou B.R., Xu Y., Guo S.L., Xu Y., Wang Y., Zhu F., Permatasari F., Wu D., Yin Z.Q., and Luo D, The effect of conditioned media of adipose-derived stem cells on wound healing after ablative fractional carbon dioxide laser resurfacing, Biomed. Res. Int., 2013 (2013) [3] Kim, W.S., Park B.S., and Sung J.H., The wound-healing and antioxidant effects of adipose-derived stem cells, Expert Opin. Biol. Ther., 9 (2009) [4] Alexaki, V.I., Simantiraki, D., Panayiotopoulou, M., Rasouli, O., Venihaki, M., Castana, O., Alexakis, D., Kampa, M., Stathopoulos, E.N., and Castanas, E., Adipose tissue-derived mesenchymal cells support skin reepithelialization through secretion of KGF-1 and PDGF- BB: comparison with dermal fibroblasts, Cell Transplant, 21 (2012) [5] Werner, S., Smola, H., Liao, X., Longaker, M.T., Krieg, T., Hofschneider, P.H., and Williams, L.T., The function of KGF in morphogenesis of epithelium and reepithelialization of wounds, Science, 266 (1994) [6] Beer, H.D., Gassmann, M.G., Munz, B., Steiling, H., Engelhardt, F., Bleuel, K., and Werner, S., Expression and function of keratinocyte growth factor and activin in skin morphogenesis and cutaneous wound repair, J. Investig. Dermatol. Symp. Proc., 5 (2000) [7] Birsoy, K., Chen, Z., and Friedman, J., Transcriptional regulation of adipogenesis by KLF4, Cell Metab., 7 (2008) [8] Dey, B.K., Stalker, L., Schnerch, A., Bhatia, M., Taylor-Papidimitriou, J., and Wynder, C., The histone demethylase KDM5b/JARID1b plays a role in cell fate decisions by blocking terminal differentiation, Mol. Cell. Biol., 28 (2008) [9] Gasbarrini, A., Gene profiling of bone narrow- and adipose tissue-derived stromal cells: a key role of Kruppel-like factor 4 in cell fate regulation, Cytotherapy, 13 (2011) [10] Nebbioso, A., Dell Aversana, C., Bugge, A., Sarno, R., Valente, S., Rotili, D., Manzo, F., Teti, D., Mandrup, S., Ciana, P., Maggi, A., Mai, A., Gronemeyer, H., and Altucci, L., HDACs class II-selective inhibition alters nuclear receptor-dependent differentiation, J. Mol. Endocrinol., 45 (2010) [11] Yoo, E.J., Chung, J.J., Choe, S.S., Kim, K.H., and Kim, J.B., Down-regulation of histone deacetylases stimulates adipocyte differentiation, J. Biol. Chem., 281 (2006) [12] Adams, I.R., and McLaren, A., Identification and characterization of mrif1: a mouse telomere-associated protein highly expressed in germ cells and embryo-derived pluripotent stem cells, Dev. Dyn., 229 (2004) [13] Zhang, T., Guan, H., and Yang, K., Keratinocyte growth factor promotes preadipocyte proliferation via an autocrine mechanism, J. Cell. Biochem., 109 (2010) Corresponding author IFSCC Magazine

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23 A Psychophysiological Approach to Substantiate Efficacy of Bath Additives Ralf Stürmer 1, Jürgen Blaak 2, Mareile Opwis 3, Jennifer Schmidt 4, Peter Staib 2, Rainer Wohlfart 2, Wolfram Boucsein 5 1 psyrecon Institute for Psychophysiological Research, Wuppertal, Germany 2 Kneipp GmbH, Research & Development, Würzburg, Germany 3 University of Hagen, Department of Health Psychology, Hagen, Germany 4 University of Wuppertal, Department of Clinical Psychology and Psychotherapy, Wuppertal, Germany 5 Deceased Keywords: Claim support, efficacy testing, bathing, psychophysiology, aromacology ABSTRACT Bath additives are used for inducing certain dermatological effects on the skin surface or aromacological effects on mood, emotion and well-being. Ongoing development of objective measurements and methods to obtain deeper aromatherapeutic insights and provide substantial support for cosmetic product efficacy is necessary. Moreover, laboratory studies with olfactory stimuli using objective measures of physiological changes are sparse, especially those recording autonomic nervous system changes. The INTRODUCTION Scents of bath additives can be regarded as constituting the most important factor for inducing relaxation or arousal and positive emotional states while taking a bath besides the cardiovascular-mediated relaxation that may result from the body being immersed in warm water. Based on previous, unpublished findings with two bath additive scents demonstrating their stimulating vs. relaxing properties, the present study was undertaken two additives in an attempt to prove the corresponding effects when taking a real bath by comparison with a control condition. The application of scents emanating from herbs, flowers and other plant parts to humans is often subsumed under the term aromatherapy, the primary aim of which is the treatment of various diseases [1]. Distinctive scents of certain plants are normally carried in ethereal oils, also called essential oils. Except for research activities focusing on their possible therapeutic applications, which in most cases do not adhere to scientific standards [2], more general studies investigating the effect of essential oils on arousal and/or mood are sparse. Especially the relaxing/ calming properties of lavender are assumed to have been demonstrated sufficiently, both in mice [3] and humans [4]. On the other present study investigated aromatherapeutic effects of bath additives while actually taking a bath, comparing an arousing and a relaxing additive with a control condition during a 20-minute bath and afterwards. Thirty female participants volunteered for this experiment, which was performed as a withinsubject design. Subjective evaluations using a standardized questionnaire were combined with objective psychophysiological measurements. The subjective ratings indicated significant changes in mood and self-perceived arousal in favor of the hand, the essential oil of jasmine is considered to have stimulating effects [5]. General arousing effects have also been observed for limonene [6], whereas sandalwood [7], chamomile [8] and incense [9] have shown relaxing effects as well. specific purpose of the additive, as shown by psychophysiological responses that identified significant bodily relaxation or arousal for the particular additive compared with the response in the control condition. Our results show that psychophysiological measurements can be used for a significant differentiation between bath additives under investigation, even while actually taking a bath. Furthermore, the novel multivariate psychophysiological approach used here might be a suitable method for bath product evaluation, e.g., claim substantiation. The use of certain scents in cosmetics and other lifestyle products such as bath additives is not due directly to their suggested therapeutic properties. However, these are often taken as background information for the scents expected effect on well-being. Besides subjective reports, the scientific value of which may be not easy to determine, objective noninvasive psychophysiological measurements can be used to record the effect of certain scents on arousal and/or cognitive and emotional states in humans. Arousal can be quantified by measuring the effects on the central nervous system using the electroencephalogram (EEG). By means of EEG recordings, Yagu [10] confirmed the above-mentioned opposite effects of lavender and jasmine, since inhalation of lavender reduced fast EEG activity, whereas inhalation of jasmine diminished slow EEG activity. Effects of scents on brain wave patterns were also found by Lorig and Schwartz [11]. A number of studies used the EEG-derived contingent negative variation (CNV) for probing psychophysiological effects of scents in humans. In 1988, Torii et al. compared the sedating and stimulating effects of altogether 20 essential oils by means of an EEG-based stimulus-elicited averaging technique. They found that lavender significantly reduced the CNV magnitude, which was in turn significantly increased by jasmine, thus objectively confirming their stimulating and relaxing effects, respectively [12]. These effects of lavender and jasmine on the CNV were also described and confirmed in later years [13-14]. Since measuring the CNV requires at least 20 iterations of repeated stimulation with the same scent, this method will not be applicable to a real bath session. Therefore, we will restrict ourselves to spontaneous EEG activity. Interestingly, Lis-Balchin [15] reported few non-eeg related psychophysiological results for humans in her handbook on Aromatherapy Science. However, since scents of essential oils are believed to exert their effects on emotion and well-being via the limbic system [16], psychophysiological measurements related to the autonomic nervous system should have been the first choice for objectively determining their effects on relaxation, arousal and emotion. The majority of studies investigating the responses of the autonomic nervous system to scents considered the electrodermal activity and heart rate. The heart rate normally decreases when humans smell pleasant scents, whereas unpleasant IFSCC Magazine

24 scents evoke an increase in heart rate [17, 18]. Using electrodermal activity, which constitutes a primary measure of emotion in the autonomic nervous system [19], it could be demonstrated that the skin conductance responses (SCR) can be modulated by perception of a scent [20-22, 7]. More precisely, variations in SCR amplitudes were found to correspond to certain properties associated with the probed scents like concentration or subjectively rated intensity and pleasantness. Scents with weak intensities gave lower SCR amplitudes than those with higher intensities. Similar results have been found for the relationship between intensity ratings and arousal. In general, negatively experienced scents gave higher SCR amplitudes than scents that were experienced as pleasant. However, the appropriate results might be confounded with the perceived intensity of scents differing in valence. Bensafi et al. showed that SCR amplitudes correlated both with rated unpleasantness and intensity of scents [18]. Furthermore, they found a weak but significant correlation between SCR amplitudes and unfamiliarity of scents, indicating additional influencing factors. In line with this, studies performed by Moskowitz and colleagues suggested that the relationships between intensity (i.e., concentration) of scents and pleasantness are rather complex and depend on the specific scents investigated [23, 24]. Parameters related to the autonomous nervous system other than electrodermal activity, heart rate or additional cardiovascular measurements were seldom used to investigate physiological changes in response to olfactory stimulation. There were reports using pupil dilation [25], skin blood flow [26], and skin temperature [6]. Somatomotor responses were investigated as muscle tension [27, 28] or eye blink magnitude [29]. The psychophysiological approach used for the present study comprised a wide spectrum of central and autonomic nervous system, somatomotor and subjective measurements. Based on previous findings obtained with scents of cosmetics products, we applied a comprehensive methodology called Objective Emotional Assessment (OEA), which was developed and refined during the last decade at the University of Wuppertal, Germany [30]. By combining continuously recording of heart rate, heart rate variability (HRV), electrodermal activity and pulse volume amplitude (PVA) with facial electromyogram (EMG) and subjective ratings, OEA permits a fine-grained analysis of responses in a two-dimensional system made up of arousal and emotional valence. Psychophysiological measurements before, during and after the presentation of distinct stimuli such as scents together with subjective ratings of arousal, emotion and certain features of the scentcarrying cosmetic product are subjected to a discriminant analysis. This allows calculation of statistical significances for the observed differences between the centroids that have been demonstrated corresponding to the probed scents [31]. An earlier, yet unpublished study from our laboratory with a pure olfactory presentation of different scents used for bath additives manufactured by Kneipp included an essential oil labeled Joy of Life ( Lebensfreude ) containing stimulating components such as Litsea cubeba and citrus oils. Results obtained by means of OEA revealed that the scent of Joy of Life induced activation together with positive valence. The aim of the present study was to confirm these effects during an actual bath in a bath tub. A further aim was to compare this activating additive with a newly developed relaxing scent labeled Deep Relaxation ( Tiefenentspannung ) which contains balsamic components such as Patchouli oil and Siam benzoin. In addition to the classical OEA measurements, we recorded the EEG to our results comparable with those from EEG research on scents. However, the EEG data will not be presented in this paper, which restricts itself to our OEA methodology. Our hypotheses based on the presumed effects of the essential oils contained in the bath additives were that Joy of Life would increase both activation and positive mood, whereas Deep Relaxation would increase relaxation and calmness without making the participants tired or exhausted. The results with these additives were compared with those of a control condition, i.e. bathing without an additive. To the best of our knowledge, this is the first investigation of bath additive scents under the naturalistic condition of taking a bath with most parts of the body immersed under water. Earlier studies used only foot baths [4]. It should be noted that the two bath additives investigated here were different colors ( Joy of Life : yellow, Deep Relaxation : blue). However, since our comparison was focused on the bath additives as sold to consumers, an additional experimental variation of colors was not attempted. EXPERIMENTAL Subjects and study protocol Thirty female participants (19 55 years, mean age 25.1) volunteered for the study. Female participants were chosen because their olfactory sensitivity is generally better than those of males [32], which may be due, however, more to a cognitive rather than a perceptive difference between genders [33]. Persons with skin diseases or olfactory problems were excluded from the study. Since the results concerning general changes in olfactory sensitivity during the menstrual cycle and/or as a consequence of taking oral contraceptives are not unequivocal [34-36], participants were not asked for their respective data. The experiment was performed as a within-subject design, comparing the two bath additives, Joy of Life and Deep Relaxation, each composed of different essential oils, and a condition without additive with each other. The ingredients of Joy of Life are: Polysorbate 20, Aqua, Glycerin, 5% perfume (a complex composition with essential oils, including Litsea Cubeba Fruit Oil, Citrus Medica Limonum Peel Oil, Cymbopogon Nardus Oil), Helianthus Annuus Seed Oil, Tocopherol, CI47005 and CI The Joy of Life perfume oil was developed using empirical knowledge of aromatherapy. It has a very light, sparkling, fresh note that gets its lively character from the citral in Litsea cubeba oil, which is rounded out by citrus oils from lemons and oranges (main component limonene) and a trace of citronella oil (characterized by its citronellal content). The bath additive Deep Relaxation ingredients are: Polysorbate 20, Aqua, Glycerin, 6% perfume (a complex composition with essential oils, including Pogostemon Cablin Oil, Styrax Tonkinensis Resin Extract), Sodium Levulinate, Sodium Anisate, Citric Acid, Tocopherol, CI28440 and CI Deep Relaxation has a very heavy balsamwoody note with earthy undertones based on a composition of patchouli oil, Siam benzoin, vanilla and sandal odorants. The Deep Relaxation perfume oil also was developed using empirical knowledge of aromatherapy. It was inspired by the agarwood used in the ancient world, which was also referred to as oud. Comparable fragrance notes have been used by shamans of all cultures since ancient times for meditative purposes. Twenty milliliters of the respective bath additive were added to the water in a bathtub containing approximately 140 liters. The sessions took place on weekdays either at 10 a.m. or 1 p.m. The hotel room and bathroom were cleaned and equipped with fresh towels by the hotel staff before each session. The 24 IFSCC Magazine

25 three experimental conditions taking a bath with the additive Joy of Life, with the additive Deep Relaxation and under the control conditions were alternated over the three sessions to prevent serial effects. Psychophysiological measurements were performed in the bathtub of a hotel room under standardized conditions (room temperature C). Each participant appeared three weeks in a row on the same weekday at the same time of day for a bath. Participants wore a bikini or a bathing suit. All participants were female. Written informed consent was obtained prior to the first session after briefing the participants about the objective of the study and the nature of the psychophysiological measures. Furthermore, the participants filled in a questionnaire on their general demographic data, cigarette consumption, skin type, frequency of taking a bath and amount spent monthly on body care products. After 10 minutes of initial baseline measurements, the participants were placed in the bathtub for 20 minutes after adapting the original 37 C water to the individual comfort level, if necessary. The water temperature was controlled regularly and did not decrease more than 1 C over the 20 minutes. Afterwards the participants covered their body with a towel and answered the mood questionnaire plus several evaluative questions on the bath additive. Physiological recordings were taken by means of a Varioport ambulatory monitoring system (Becker Meditec, Karlsruhe, Germany). Electrodermal activity was recorded with 0.8 cm diameter Ag/AgCl electrodes and isotonic electrolyte jelly from right-hand thenar/ hypothenar sites with 0.5 V constant voltage as the skin conductance (SC) using a sampling rate of 64 Hz and a 0.5 Hz filter and 0.01 microsiemens as the criteria for amplitudes. The hand with the electrodermal activity electrodes was kept out of the water for the whole time of the bath. To prevent the electrodes for recording the heart rate from being immersed in water, a modified Einthoven- 1 recording was used (right clavicle vs. left upper arm with grounding on the sternum) with a sampling rate of 256 Hz and a 15-Hz filter. In addition to the psychophysiological measures, a standardized 24-item mood questionnaire [37] was filled in by the participants before and after the bath. The psychophysiological recordings were parameterized by means of our customized computer software as follows: From the skin conductance signal, the mean level (SCL), the number of nonspecific skin conductance responses (NS.SCR freq.) and their mean amplitude (NS.SCR amp.) were calculated for 5- minutes segments for which heart rate was also obtained from the modified Einthoven-1 lead described above. In addition to heart rate (in beats per minute), the heart rate variability (HRV) was calculated as the standard deviation (SD) and as the mean root mean square of successive differences (RMSSD) in the 5-minute segments. The scores for the 2 x 5 minutes segments of the initial baseline were averaged and subtracted from the response scores obtained during the bathing procedure to correct for possible baseline effects resulting from individual differences or different times of day (10 a.m. vs. 1 p.m.). Statistical analysis Both univariate and multivariate statistical evaluations were performed, comparing the three experimental conditions Joy of Life, Deep Relaxation, and Control for each mood questionnaire item and the physiological parameters. Since the sample size was below 50, a Shapiro-Wilk test of normal distribution was performed, revealing that more than 90% of the data were not distributed normally. Therefore, nonparametric tests were applied for each single mood questionnaire item, i.e., Friedman (analysis of variance) tests for overall effects with subsequent Wilcoxon paired comparison tests. In addition, all subjective and physiological measurements were subjected together to a multivariate analysis by means of discriminant analysis. For this purpose, the physiological measurements were initial-baseline corrected and evaluated for the following segments: 1 5, 6 10, 11 15, and minutes during the bath, as well as 1 5 and 6 10 minutes of the after-bath baseline. To fulfill the requirement of independent samples, the portion of variance owing to the different sequences of bath conditions were eliminated by means of a matrix transformation using a procedure that has been approved by Tabachnick and Fidell [38]. Wilk s-lambda and Chi-square test values were used for determining the significance of the discriminant functions, and the factor loadings for each variable, obtained as standardized canonical discriminant function coefficient weights, were used for interpreting the discriminant functions. Finally, the distances between group centroids for the three experimental conditions were tested pairwise for significance. Probabilities exceeding a P = 0.05 (two-tailed) level of significance were marked with one asterisk, those exceeding P = 0.01 with two asterisks. Another Chi-square tests was performed for the psychophysiological parameters, comparing the number of participants who showed an increase or decrease of more than 10% in all electrodermal and heart-activity related parameters during the bath and the afterbath baseline compared with the initial baseline, and those who did not. For this purpose, we averaged the recording sections: minutes 1 10 of the bath, minutes of the bath, 10 minutes initial baseline and 10 minutes after-bath baseline. RESULTS The present paper will be restricted to the results from the classical OEA, i.e., subjective and autonomic nervous system-related psychophysiological measures. Because most of the literature on the effects of scents refers to subjective measures, those will be presented first followed by the psychophysiological results and finally the results from the multivariate evaluation, which included both the subjective and physiological measures. Subjective measurements For evaluation the mood changes during the bathing procedure, the items of the mood questionnaire obtained after the bath were compared with those before taking the bath by means of Wilcoxon tests. Table I shows those items showing significant differences or a tendency towards significant differences (P < 0.10) separately for the three experimental conditions (i.e., bathing with Joy of Life, Deep Relaxation, or without additive = control). As Table I shows, there was no significant mood change in the control condition without additive except for a tendency to become sleepier. Taking a bath with Joy of Life made the participants feel better and fresher, with the difference being highly significant, but it also made them feel significantly less restless and more rested and less tired, uneasy, displeased and tense with a tendency to feel less nervous and droopy. Furthermore, they felt significantly more vivid, comfortable, balanced and happy, but also more exhausted, which may have been more on the relaxed side (P < 0.10). Taking a bath with Deep Relaxation left the participants feeling significantly more rested, composed, relaxed, balanced and calm and significantly less tense. They further showed a significant decrease in restlessness and felt significantly better, with a tendency to become more content but also sleepier. Table II shows the differences between the three experimental conditions in the nine initial-baseline-corrected after-bath baseline subjective items from the mood question- IFSCC Magazine

26 Table I: Mean and standard deviation (SD) for those items of the mood questionnaire for which changes from the initial baseline to the after-bath baseline reached significance in the Wilcoxon test listed separately for the three experimental conditions. CON = control condition; JOL = Joy of Life ; DRL = Deep Relaxation ; sig. = significance; *P < 0.05; **P < 0.01 (two-tailed). Table II: Friedman test results (Chi-square), mean (M) and standard deviation (SD), and post-hoc Wilcoxon paired comparison test results for the mood questionnaire items showing significant differences between the after-bath and the initial baseline. CON = control condition; JOL = Joy of Life ; DRL = Deep Relaxation ; *P < 0.05; **P < 0.01 (two-tailed). naire that reach significance in the Friedman test. As can be inferred from Table II, participants rated themselves as highly significantly less exhausted and droopy (P < 0.01) and significantly less tired, but highly significantly more vivid and fresh after taking a bath with Joy of Life compared with the control, as well as highly significantly fresher and significantly less tired and relaxed than with Deep Relaxation. On the other hand, after taking a bath with Deep Relaxation they rated themselves highly significantly more rested, composed and relaxed and significantly more balanced than with the control as well as significantly more composed and less vivid than with Joy of Life, in addition to the above-mentioned differences between Joy of Life and Deep Relaxation. Psychophysiological measurements For the psychophysiological measures, we calculated averages for the following recording sections: minutes 1 10 of the bath, minutes of the bath, 10 minutes initial baseline and 10 minutes after-bath baseline. Chi-square tests were performed comparing the number of participants showing changes of more than 10% from their baseline means. Table III displays the Chi-square tests results for those recording sections that achieved statistical significance. To further reveal the nature of these differences, post hoc pairwise comparison Chi-square tests were performed between the different groups. All remaining comparisons gave no significant results. As can be inferred from Table III, the comparison between the first half of the bath, i.e., minutes 1 10, revealed a significant increase in the skin conductance level. Pairwise comparisons between the experimental conditions revealed significantly more participants with an increase in the skin conductance level from the control to Joy of Life and from the control to Deep Relaxation, but no significant difference between Joy of Life and Deep Relaxation. A significant increase in heart rate variability evaluated as RMSSD emerged for a group of participants during minutes 11 20, i.e., the second half of the bath. Pairwise comparisons between the experimental conditions revealed a highly significantly greater number of participants who had an increase in RMSSD from the control to Joy of Life and from the control to Deep Relaxation, but again no significant difference was observed between Joy of Life and Deep Relaxation. There was, however, also a group of participants showing a decrease in heart rate variability evaluated as RMSSD from the initial baseline to the second half of the bath, for which a tendency towards significance (P = 0.065) emerged. Pairwise comparisons revealed significantly fewer participants with RMSSD dropping from the control to Deep Relaxation and as a tendency towards significance (P = 0.067) from the control to Joy of Life. There was again no difference between Joy of Life and Deep Relaxation. In the after-bath baseline, there were significantly more participants with an increasing heart rate variability recorded as SD compared with the initial baseline. Pairwise comparisons revealed a tendency towards significance (P = 0.069) with fewer participants having an increasing SD in the control compared with Joy of Life, a highly significant difference in the same direction between Deep Relaxation and the control but no significant difference between Joy of Life and Deep Relaxation. The course of psychophysiological measures showing significant differences between the experimental conditions is shown for the skin conductance level in Figure 1 and the heart rate variability recorded as RMSSD in Figure 2. As Figure 1 shows, the skin conductance level was consistently higher in the Joy of Life condition than in the other two conditions during the entire bath. After that the skin conductance level dropped sharply, whereas the participants taking a Deep Relaxation bath showed a further increase in skin conductance level afterwards. The opposite results were obtained for the heart rate 26 IFSCC Magazine

27 variability (HRV) recorded as RMSSD, which was higher during the bath with Deep Relaxation and fell below the HRV values of Joy of Life after the bath (Figure 2). Here, the heart rate variability increased markedly for the control condition. Multivariate statistical evaluation A discriminant analysis was performed with the physiological autonomic nervous system parameters (heart rate, heart rate variability as SD and as RMSSD, skin conductance level, NS.SCR freq. and NS.SCR amp.) for the segments mentioned in the methodology section, together with the 24 items of the mood questionnaire. All physiological parameters and mood questionnaire items were baseline corrected. The two discriminant functions reached significance: Wilks-Lambda = 0.14, Chi-square = (df= 86), P < 0.01 for function 1, and Wilks-Lambda = 0.38, Chisquare = (df=42), P = 0.02 for function 2. Function 1 accounted for 52.13% and function 2 for 47.87% of the total variance. Factor loadings (i.e., standardized canonical discriminant function coefficients) for the variables contributing to the discriminant functions with significances of at least P = 0.10 (positive or negative) are reported in Table IV for function 1 and 2. Eighty-nine percent of the cases were correctly re-classified. Using the factor loadings from Table IV, the positive and negative extremes of discriminant functions 1 and 2 were labelled as shown in Figure 3. This figure depicts also the centroids for the three experimental conditions, i.e., the two bath additives and the control condition without additive. All differences between the three group centroids were statistically significant: the control vs. Joy of Life (F43/45 = 1.85, P = 0.02), the control vs. Deep Relaxation (F43/45 =1.8, P = 0.03) and Joy of Life vs. Deep Relaxation (F43/45 =1.72, P = 0.04). For the first time, effects of bath additives have been objectively investigated under the naturalistic condition of actually taking a bath with immersion of the whole body in water. For this purpose, we chose a psychophysiological approach called OEA. The usability of OEA, which had been shown repeatedly in studies with cosmetics, was convincingly demonstrated in the present study for determining the effects of bath additives on emotion, arousal, and well-being. We compared two different additives with compositions of scents, Joy of Life and Deep Relaxation, and a control condition without an additive in a within-subjects design using female participants. Various significant differences between each of the two bath additives and the control condition were obtained by means of a mood questionnaire (Table II). Joy of Life made the participants feel more vivid and fresh as well as less exhausted or tired compared with the control condition without additive, whereas Deep Relaxation made them feeling more relaxed, composed and balanced. The differences between Joy of Life and Deep Relaxation were also signifi- Table III: Chi-square test results of the group differences for participants who had changes more than 10% from baseline in their physiological parameters and post-hoc pairwise comparisons. SCL = skin conductance level; HRV = heart rate variability; RMSSD = root mean square of successive difference; HR = heart rate; CON = control condition; JOL = Joy of Life ; DRL = Deep Relaxation ; sig. = significance; = Parameter rising more than 10% from the initial baseline; = Parameter dropping more than 10 % from the initial baseline. DISCUSSION Table IV: Standardized canonical discriminant function coefficients for the two discriminant functions showing at least a value of 0.10 in both directions in either function 1 or 2 (marked as bold). The items of the 24-item mood questionnaire and the physiological measures were ordered by decreasing discriminant powers for positive coefficients, followed by increasing discriminant powers for negative coefficients for function 1. NS.SCR amp. = nonspecific skin conductance response amplitude; SCL = skin conductance level; RMSSD = root mean square of successive difference; HR = heart rate. IFSCC Magazine

28 cant, with the participants feeling fresher, more vivid and less tired after the bath with Joy of Life, whereas Deep Relaxation induced more subjective relaxation and feelings of composure than Joy of Life. An additional analysis of the questionnaire results revealed that the changes in mood ratings from before to after taking a bath differed not only between conditions but also within conditions (Table I). Interestingly, bathing without additive did not induce much of a change in mood ratings, except for a tendency to become sleepier. The differential effects of the two bath additives found in the subjective domain complement those obtained by the objective physiological measures. Throughout the whole bath the skin conductance level was higher in the Joy of Life condition than in the other conditions (Figure 1), with the difference being significant during the first 10 minutes compared with the condition without additive. Since an increased skin conductance can be regarded as an objective indicator for general arousal [39], which was also demonstrated for smelling pure odors by Bensafi et al. [18], the arousing effect of Joy of Life seen in the mood questionnaire could be confirmed objectively. The difference between Deep Relaxation and the control was statistically significant, although the mean skin conductance level was not as high in the Deep Relaxation as in the Joy of Life condition. This could be a short-time arousing effect of bathing with unknown additives. This effect vanished during the second half of the bath. Here, the participants in the Deep Relaxation condition had the lowest skin conductance level. However, as can be inferred from Figure 1, after taking a bath with Deep Relaxation participants were more aroused than after the other conditions, albeit not significantly. This can be interpreted as a rebound effect after emerging from the relaxing bath. The positive-valence arousal effect of Joy of Life, which was demonstrated both subjectively and objectively, persisted after the participants had emerged from the bath. In contrast, the heart rate variability (recorded as RMSSD) was highest in the Deep Relaxation condition (Figure 2), reaching significance during the second half of the bath compared with the two other conditions. An increase in heart rate variability constitutes an objective sign of relaxation [41]. In addition, the HRV results showed the highest number of participants with heart rate variability dropping in the control condition. Therefore, the relaxing effect of Deep Relaxation determined in the mood questionnaire was also confirmed by means of Figure 1: Course of the skin conductance level over the bath and after-bath 5-minute segments for the three experimental conditions. CON = control condition; JOL = Joy of Life ; DRL = Deep Relaxation ; SCL = skin conductance level. Figure 2: Course of the heart rate variability recorded as the RMSSD over the bath and after bath 5-minute segments for the three experimental conditions. CON = control condition; JOL = Joy of Life ; DRL = Deep Relaxation ; HRV = heart rate variability; RMSSD = root mean square of successive difference. this objective psychophysiological measure. Interestingly, Figure 2 displays a sharp increase in heart rate variability after emerging from the bath without additive (the control), which supports what was seen in the mood questionnaire, i.e., that taking a bath without additive was not really comfortable so that finishing it might have been a relief. The use of bath additives, especially Deep Relaxation, prevented such a sudden change in psychophysiological responses, since heart rate variability continued to decrease steadily, which was most pronounced in the Deep Relaxation condition. Therefore, Deep Relaxation can be regarded as a subjectively and objectively relaxing bath additive with smoothly emerging aftereffects. The heart rate variability recorded as SD showed a significantly higher number of participants who had an increase in the after-bath baseline (Table III). This relaxation effect is probably due to the feeling of relief that participants 28 IFSCC Magazine

29 experienced when they emerged from the rather boring bath situation. The ultimate probe of our OEA approach is to obtain significant distances between the probed conditions by combining subjective and objective measures of arousal, emotion and well-being in a single discriminant analysis. As Figure 3 shows, such an approach was successful in the case of the bath additives compared in the present study. The positive extreme of discriminant function 1 which separates the two bath additives from the control condition is characterized by the participants feeling fresh, awake, happy and composed. Loadings of Joy of Life were markedly higher in this factor than those of Deep Relaxation. In addition to the subjectively reported effects, an enhanced NS.SCR amp. was observed during all time periods (except for the minutes segment) while taking a bath with Joy of Life. This cannot be regarded as an indicator for mere general arousal but for behavior-directed activation [39]. This kind of activation, which indicates an increased readiness for somatomotor actions, persisted during the afterbath baseline. As early as in the first five minutes of the Joy of Life bath experience, participants showed an increase in both NS.SCR freq. and skin conductance level, which further supports the physiologically activating effect of Joy of Life, albeit in a more general arousing manner. The positive extreme of discriminant function 2 which separates Deep Relaxation from the other conditions is characterized by the items composed, calm and relaxed. However, the items tired and unhappy also held high loadings at this extreme of the function. Probably, our participants were excited when first confronted with the unfamiliar stimulus of the Deep Relaxation bath. This is mirrored in the fact that during the first five minutes of the bath the heart rate loaded high on this function. Still, participants showed evidence of factual relaxation at the same time (RMSSD in the first five minutes of the bath). Another distinctive feature is found in a short-term increase in several parameters of activation immediately after the bath (skin conductance level, heart rate, NS.SCR amp. during minutes 1 5 after the bath). It can be inferred that our participants might have been very relaxed during the bath with Deep Relaxation and were slightly stressed by being withdrawn from the pleasant setting. This might also explain the loadings of the item unhappy at this extreme of the function. We observed as a main difference between the control and the two additive conditions (especially Joy of Life ) that participants felt rather exhausted, droopy, tired, but also restless after bathing without an additive. In contrast, after the bath session with the control was finished, they showed higher heart rate variability during the after-bath baseline as an index of bodily relaxation (RMSSD and SD during minutes 1 5 min, SD during minutes 6 10). As mentioned above, the participants started to be relaxed after leaving the rather boring bath situation. Taking a bath without additive might have even induced a negatively toned, stress-like response, as shown by heart rate acceleration during minutes of the bath. CONCLUSION Our study revealed the proposed effects of the tested bath additives, thereby ensuring the possibility to use the present methodology for cosmetic claim substantiation. Since the effects may be influenced by selected essential oils, in the future it would be interesting to dissect the impact of specific fragrances and/or molecules using our approach. Finally, the multivariate psychophysiological approach used here might be a new suitable method for performing fine-grained analyses in the field of bath product evaluation and cosmetic claim substantiation. ACKNOWLEDGEMENTS The present work is dedicated to Wolfram Boucsein, former Professor of Physiological Psychology at the University of Wuppertal, Germany and founder of the psyrecon Institute for Psychophysiological Research. This research was funded by Kneipp GmbH, Würzburg, Germany. In addition, the test products were developed and provided by Kneipp. REFERENCES [1] Cooke, B., and Ernst, E., Aromatherapy: a systematic review, Brit. J. Gen. Pract., 50 (2000) [2] Martin, G. N., Olfactory remediation: Current evidence and possible applications, Soc. Sci. Med., 43 (1996) [3] Buchbauer, G., Jirovetz, L., Jäger, W., Plank, C., and Dietrich, H., Fragrance compounds and essential oils with sedative effects upon inhalation, J. Pharm. 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31 From an Eco-Friendly To an Eco-Improving World of Cosmetic Science Addressing Water Pollution in Emerging and Developing Societies Chaikriangkrai Amata KOSÉ Corporation, Japan Winner of the IFSCC MG de Navarre Young Scientist Prize, 2015 Imagine what would happen if cosmetic products were not only eco-friendly but also improved the environment in which we live. For instance, if our most popular shampoos or conditioners contained components that improved the quality of water, every time we washed those components would enter our water systems and begin to tackle the water pollution that our society has caused through rapid population growth and poor wastewater management. In emerging and developing countries, water pollution and other environmental problems are becoming increasingly a concern. Without an obvious solution in sight, perhaps cosmetic science can offer an answer. Billions of people around the world use cosmetic products every day. Cosmetic products play an essential role in everyone s lives, providing some with improved health and cleanliness and others with greater psychological comfort. For example, some people use soap to clean their bodies, while others use the aromatic scent of soaps to relax after a hard day's work. Until now, cosmetic scientists have focused on developing new products that have improved functions, fewer risks, more pleasant aromas, and more affordable prices. We see the results of this work in better human hygiene and health, improved physical appearance, and greater personal selfesteem, which ultimately increase people's chances in society. The social effects resulting from the advancement of cosmetic sciences can be seen in the following chart (Figure 1). However, using more cosmetic products means producing more waste, which in turn leads to greater pollution of the environment. Although cosmetic companies have strived to reduce waste by using refillable or self-degradable packaging materials, the massive growth of the cosmetics industry means that we need to consider further the impact of our products on the environment around us. Every day around the world cosmetic products are rinsed and washed away together with wastewater. In developed countries the wastewater released from households is usually precipitated, treated by many chemical and biological processes, and then released relatively safely into the environment. On the other hand, wastewater management in emerging and developing countries is still mostly lacking. In many rural districts and even in parts of large Figure 1: Social effects resulting from the advancement of cosmetic science cities, such as slum areas, the deficiencies in wastewater management directly impact the human hygiene of people in those areas and in the surrounding districts. For example, according to data from The Center for Strategic and International Studies (CSIS), more than 1.38 million people in China were diagnosed in 2012 with viral hepatitis, a disease that can be contracted by drinking contaminated water or eating contaminated food [1]. In India river water polluted by untreated water is used for drinking, bathing, and washing. Data reported by E. Brainerd and N. Menon shows that in the period when agriculture was rapidly expanding and fertilizer was being heavily used, there was a higher infant mortality and neonatal mortality than in periods with less agriculture activity [2]. Poor wastewater management is not the only explanation for water pollution in emerging and developing societies. Another root cause is the problem of overpopulation. According to the United Nations (2014), the population of China, India, and Indonesia are 1.39 billion, 1.27 billion, 0.25 billion, respectively, and these figures are increasing every year by approximately 1%. The forecast for populations of these countries in 2020 is 1.43 billion, 1.35 billion and 0.27 billion, respectively [3]. If these numbers are correct, we can expect even more people to use cosmetics, and indeed, even more waste and pollution are likely to be generated and released into the environment. Euromonitor (May 2015), for example, has shown that the market size of beauty and personal care products in China increased more than 7% every year from 2009 to 2014, and a similar trend can be seen in other emerging and developing countries [4] (see Figure 2). If we do nothing, the IFSCC Magazine

32 Figure 2: Market size of cosmetic products in emerging and developing countries Figure 3: Impact of eco-friendly and ecoimproving cosmetics on water pollution continuing problems of rapid overpopulation are likely to intensify the effects of water pollution in these societies. One way to protect the world and the water environment is to consider how we can reduce the amounts of environmentally unfriendly ingredients in cosmetics. Bath shampoos and washes contain many types of detergents, including phosphatecontaining, nitrate-containing, and sulfatecontaining detergents. Phosphates and nitrates support the growth of plants, including algae, while sulfates are bioaccumulative and toxic to marine life. Excessive amounts of algae may lead directly to poor water quality. This is why the environmental impact from these cosmetic ingredients should be more seriously considered. Currently, many researchers are attempting to design products with fewer of these environmentally unfriendly ingredients, for example, by using surfactant-free emulsions and sulfate-free body cleansers. However, the reduction or removal of such detergents or surfactants generally results in an unpleasant cleansing function or emulsion breakage. Therefore, we need to focus more on the development of products that maintain the excellent functions of current products but have less of a negative impact on the environment in which we live. Next, we need to focus on the development Figure 4: Cosmetic solution to water pollution resulting from big-city overpopulation of substituted ingredients which are also eco-friendly. Usually, the development of cosmetic ingredients starts with finding substances that have the required functions. Next, these substances are analyzed and the resulting data are evaluated to determine the substances' health safety, chemical stability, and physical properties. As a result of such work, in past decades cosmetic scientists have managed to develop many substituted ingredients, such as amino acid based detergents, that have lower skin irritation and are more ecofriendly than products containing phosphates or sulfates. On the other hand, the environmental impact of such ingredients is usually neglected. Even though there are some non-profit organizations dealing with this issue, such as The Environmental Working Group (EWG), the effects of cosmetic ingredients on the environment are still of far less concern than their impact on direct human health and safety. Reduction and replacement of environmentally unfriendly cosmetic ingredients are likely to slow water pollution, especially in emerging and developing countries. However, we can also predict that numerous other causes of pollution, such as the effects of the mining industry, the use of chemicals in agriculture, and even radioactive wastewater, will mean that more direct measures must be taken, as shown in Figure 3. One direct way to address water pollution in society today, particularly in emerging and developing countries, is to create a new role for cosmetics as an environment improver. If we can create novel cosmetic products that directly address the unique and complex water pollution issues in each area, we can create a cleaner, brighter, and more sustainable society and perhaps even new markets for our cosmetic products. As an example, water pollution in coastal areas of big cities is generally caused by an excessive drainage of phosphates and nitrates into the ocean. In 2013, an enormous 28,900 square km algal bloom appeared in the Yellow Sea of China, which was considered by many to be related to pollution from phosphates and nitrates discharged from agricultural production. Even though algae are not toxic to humans or animals, their presence can block sunlight from entering the sea, directly affecting marine life and water quality. Reports have shown that the growth of algae can be inhibited by controlling the amount of Fe ions, a trace element used for the algae s photosynthesis [5]. Therefore, we might consider adding to cosmetic products Fe chelating agents that later flow into the sea, slowing down the growth rate of algae and consequently improving the quality of water. Through cosmetic science research we can determine the most appropriate chelating agent, find the percentage of agent needed to improve water quality, and of course, ensure that the product is safe for human use. If these tests are successful, we can then consider adding an appropriate amount of the chelating agents to products, such as body soaps and shampoos, and develop an action plan such as marketing the products in coastal areas where water pollution is a major concern. This idea is illustrated in Figure 4. Water quality problems can also emerge as a result of completely different chemical mechanisms. Countries like Peru, Ecuador and Colombia, for example, are situated along the equatorial Pacific Ocean, where the amounts of phosphates and nitrates in the water are in significant excess, whereas the concentration of free Fe ions is insufficient. This imbalance results in a highnutrient, low-chlorophyll (HNLC) environment in which phytoplankton is generally not able to survive. When the water in HNLC areas is polluted even just by biodecomposable pollutants, the water quality can easily worsen due to a lack of phytoplankton. Coastal HNLC areas are especially affected because large amounts of pollutants that are emitted from industry cannot be adequately decomposed by phytoplankton [6]. 32 IFSCC Magazine

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