CO S MET IC S I N F L A MMAT IO N H. Chajra, D. Auriol, K. Schweikert, F. Lefevre* Targeting Inflammatory Pathways to Reduce Dark Circles and Puffiness ■ Introduction Dark circles under or around the eyes (characterized by the darkening of the eyelids and periorbital skin) are a common cosmetic problem for a large number of people as is often associated with tiredness, stressed or aged appearance (1). Various causes can lead to the ap- pearance of dark circles around the eyes, including aging with increased skin laxity, post-inflammatory hyperpigmentation secondary to atopic dermatitis or allergic contact dermatitis (2, 3), or as a consequence of post-inflammatory hemodynamic congestion producing a typical bruising aspect on the lower eyelids and oedema (3). Oedema is often Abstract D ark circles are a common cosmetic problem for a large number of people. Because, most of the cosmetic products available on the market targeted either blood micro-circulation or covered the skin hyperpigmentation, we decided to develop a cosmetic ingredient, which targets the inflammatory, lymphatic and vascular origin of dark circles. In this study the efficacy of the active ingredient was assessed in vitro and on human volunteers. Our studies have shown the effectiveness of this ingredient to reduce the dark circles after 15 and 30 days of treatments. The mechanism of action demonstrated is based on both anti-inflammatory and draining properties. The active ingredient showed anti-inflammatory effects on keratinocytes by strongly inhibiting NF-KB pathway-driven IL-8 and CXCL1 releases. The inhibitory effect of the active ingredient was not mediated by a direct inhibition of NF-KB translocation but by inhibition of the NF-KB activity. This active ingredient improves the drainage function of lymphatic vessels by increasing the expression of VEGFR3 gene and controls the vascular tone by increasing the expression of Heme oxygenase-1 gene. 16 due to a malfunction of lymphatic vessels. Histo-pathological changes associated with dark circles are the increase in melanin content causing hyperpigmentation and the dilation of dermal blood vessels (4). Periocular »dark circles« fall among the most difficult complaints to address by dermatologists. There is no gold-standard treatment option. Available dermatological treatments include bleaching creams, topical retinoid acid, chemical peels, laser therapy, autologous fat transplantation, injectable fillers, surgery (blepharoplasty), and chemical peeling. Most of the cosmetic products available on the market target either skin lightening, vascular constriction (using caffeine or esculin), or they use pigments to cover the skin hyperpigmentation. Our searches targeted the inflammatory, lymphatic and vascular origins of dark circles. Gallic acid and its derivatives including epigallocatechin gallate are known for their anti-inflammatory properties by acting on the inhibition of the degranulation by neutrophils (5), decreasing cytokine production (6-9), reducing immune cell infiltration and inhibiting lipooxygenase (10). However, these molecules are well known to be either instable and/or difficult to formulate in cosmetic products due to their chemical structures. We have therefore combined in a water solution a mix of three stable and water-soluble gallic acid derivatives: gallic acid glucoside, epigallocatechin gallate glucoside, and propyl gallate (patent Libragen company EP2027279B1 (11)). We have studied in vitro the activity on the NF- B pathway of the active ingredient. More precisely SOFW-Journal | 140 | 4-2014 CO SMET ICS I NF LA MMAT I O N the effects of the active ingredient were evaluated on nuclear factor-kappa B translocation, and then on the activation of IL-8 and CXCL1 release by keratinocytes. To address the lymphatic and vascular origins, in vitro activities of the active ingredient on the lymphatic factor (VEGFR3) and the vascular factor (Heme Oxygenase -1) were studied. Finally, to evaluate the efficacy of the active ingredient to treat dark circles a clinical investigation was performed on human volunteers. ■ Materials and methods The active ingredient (trade name Unisooth EG-28) was composed by 6mM of gallic acid glucoside (CAS 131579-696), 323µM of epigallocatechin-glucoside (CAS 236072-19-7), and 117 mM propyl gallate (CAS 121-79-9). Gallic acid glucoside is the glycosylated form of gallic acid coming from oak leaves and epigallocatechin-glucoside is the glycosylated form of epigallocatechin gallate originating from green tea. Studies on the inflammatory pathways NF- B translocation in keratinocytes Cell cultures and treatments: The normal human epidermal keratinocytes (NHEK used at the 3rd passage, Bioalternatives, Gençay, France) were cultured in 96-well plates in culture medium until subconfluence at 37°C, and 5 % CO2. 20000 cells/well were seeded. The culture medium was a Keratinocyte SFM supplemented with epidermal growth factor (EGF) at 0.25 ng/mL, pituitary extract (PE) at 25 µg/ml and gentamycin at 25 µg/ml. The culture medium was then removed and replaced by assay medium containing or not the active ingredient (at 0.016, 0.08 and 0.4 %) or the references (NF- B inhibitor III at 5 µM + NF- B inhibitor V at 1 µM) and the cells were pre-incubated for 2 hours. The assay medium was a keratinocyteSFM supplemented with gentamycin at 25 µg/ml. After the pre-incubation, the cells were stimulated by the association of TNF- + IL-1 (both at 5 ng/ml) and incubated for 20 minutes. All experimental conditions were performed in triplicate. NF- B translocation- in situ immunolabeling: At the end of incubation, culture medium was discarded and the cells were washed, fixed and permeabilized. Cells were then labeled with a primary anti-p65 NF- B antibody (ref 610868; BD Biosciences, France). The primary antibody was then revealed using a fluorescent secondary antibody (GAM-Alexa 488) and the cell nuclei were labeled with Hoechst solution (bis-benzimide) in parallel. The acquisition of the images was performed with the INCell AnalyzerTM www.proDERM.de WHERE EXPERTS ARE COMMITTED INDEPENDENT CONTRACT RESEARCH DERMATOLOGY I am committed to your testing needs | OPHTHALMOLOGY | HAIR CARE | ORAL CARE APPROACH STUDY TYPES PROFESSIONAL SOLUTIONS CLAIM SUPPORT & TOLERABILITY EQUIPMENT QUALITY INNOVATIVE TECHNOLOGIES ISO 9001 CERTIFIED CONSULTING SITE MANAGEMENT +100 YEARS OF EXPERIENCE MULTICENTRE TRIALS BOOTH 1L60 and I like putting my interpersonal strengths into the recruitment of volunteers. Nicole P., Recruitment Manager at proDERM SOFW-Journal | 140 | 4-2014 17 CO S MET IC S I N F L A MMAT IO N 1000 (GE Healthcare). Controls without primary antibody were performed in order to adjust the acquisition parameters of the camera. 5 photos were taken per well for the immunolabeling of NF- B. The labeling was quantified by the measurement of the fluorescence intensity in cell nuclei (Integration of numerical data with the Developer Toolbox 1.5, GE Healthcare software). NF- B activity study in transformed human cells Cell cultures and treatments: Transformed human cells (HT29) were transfected with a NF- B reporter gene plasmid (pNiFty2-SEAP, reference: pNifty2SEAP, Invivogen, Toulouse, France). The transfected cells (500000/mL) were incubated 24 h with TNF- (20 ng/mL) in the presence or absence of active ingredient at different concentrations (from 0.01 to 4.36 %). All experimental conditions were performed in quadruplicate. Assay of NF- B activity: The contact of TNF- and transfected cells (HT29-NFB -SEAP-Cl25) induced gene expression of the NF- B pathway as well as SEAP (secreted Embryonic Alkaline Phosphatase) loaded by the plasmid pNiFty2SEAP. The expression of this gene and the production of the SEAP enzyme were directly proportional to the quantity of cytokine present in the culture media. The assay of SEAP enzyme is an indicator of NF- B pathway activation. The assay of the enzyme SEAP was performed using the Quantie blue reactif (ref repqb, Invivogen, France) according to the instructions of use provided by the fabricant. The enzyme concentration was determined by the reading of the absorbance at 650 nm. IL-8 and CXCL1 released by keratinocytes Cell cultures and treatments: The keratinocytes (NHEK) were cultured in 96-well plates in culture medium for 24 hours at 37°C, and 5 % CO2. 20000 cells/well were seeded. The culture medium was a Keratinocyte SFM supplemented with epidermal growth factor (EGF) at 0.25 ng/mL, pituitary extract (PE) at 25 µg/ ml and gentamycin at 25 µg/ml. The medium was then removed and re- 18 placed by assay medium containing or not (control) the active ingredient or the references (NF- B inhibitor III at 5 µM and dexamethasone at 10 µM) and the cells were pre-incubated for 24 hours. The assay medium was a keratinocyteSFM supplemented with gentamycin at 25 µg/ml. After the pre-incubation, the medium was removed and replaced by assay medium containing or not (control) the active ingredient or the references in presence of the association of TNF- + IL-1 (both at 5 ng/ml) and the cells were incubated for 48 hours. A nonstimulated control condition was also performed in parallel. All experimental conditions were performed in triplicate. Quantification of IL-8 and CXCL1 released by NHEK: At the end of incubation, the quantities of IL-8 (Elisa Development kit, ref 900-K18, Peprotech, Neuilly sur Seine, France) and CXCL1 (Ref. DY275, R&D Systems Europe, Lille, France) in culture supernatants were measured using ELISA kits according to the supplier’s instructions. Data management: Raw data were analyzed with Microsoft Excel software. The inter-group comparisons were performed by Student’s t-test (for paired data). The significance was judged as followed. *p <0.05; **p <0.01 and ***p< 0.001. All reported data are expressed as mean ± sem. The standard error of the mean (sem) is calculated as the standard deviation (sd) divided by the square root of sample size (sem = Sd/ n). Studies on the lymphatic and vascular markers The effects of the active ingredient at 0.04 and 0.2 % were studied on gene expression of two proteins involved in vessel functions using RT-qPCR technology. Cell cultures and treatments: Human dermal microvascular endothelial cells (HMVEC, Bioalternatives, Gençay, France) were cultured in culture medium for 48 hours (10000 cells per well). The medium was then removed and replaced with assay medium. After 24 hours of culture, medium was replaced with assay medium containing or not (control) the active ingredient then cells were incubated for 24 hours. All experimental conditions were performed in triplicate. At the end of the incubation, cells were washed in phosphate buffered saline (PBS; Life Technologies) solution and immediately frozen at -80°C until mRNA extraction. RT-qPCR and data management: Extracted mRNA from HMVEC was analyzed on a customized PCR array containing target genes (VEGFR-3 and Heme Oxygenase 1) and including 3 housekeeping genes (glyceraldehyde-3-phosphate dehydrogenase, actin beta and Ribosomal protein S28). Primer sequences used were Homo sapiens VEGFR-3 (NM_182925): sense-TGTCCTACGATGCCAGCCAGTG antisense-TTGAGGTGGTTGCCGATGTGAATG and Homo sapiens Heme Oxygenase 1 (NM_002133): sense-TCCGATGGGTCCTTACACTC antisense- ATTGCCTGGATGTGCTTTTC Total RNA was extracted using »TriPure Isolation Reagent« kit (Roche Applied Science). The amount and quality of RNA were evaluated using a lab-on-a-chip Bioanalyzer (Agilent technologies). Potential contaminant traces of genomic DNA were removed using the DNA-free system (Ambion by Life Technologies). The reverse-transcription of mRNA was conducted in presence of oligo (dT) and SuperscriptTM II reverse-transcriptase (Life Technologies). Quantification of cDNA was performed using NanoVue Plus (GE Healthcare) and adjustment of cDNA at 5 ng/µl. The PCRs (Polymerase Chain Reactions) were performed using the LightCycler® system (Roche Diagnostic, France) as described originally by Wittwer et al (12). The incorporation of fluorescence in amplified DNA was continuously measured during the PCR cycles. This resulted in a »fluorescence intensity« versus »PCR cycle« plot allowing the evaluation of a relative expression (RE) value for each marker. The RE value was expressed in arbitrary units according to the formula: 1/2number of cycles x 106 The relative expression calculated was normalized to the three housekeeping genes and to untreated cells (control). SOFW-Journal | 140 | 4-2014 CO S MET IC S I N F L A MMAT IO N Table 1 was used for the interpretation of the effect. Clinical study on human volunteers A double blind and placebo-controlled clinical evaluation was carried out on 25 volunteers (female Caucasian subjects older than 18 years) following half-face method. The active ingredient (INCI composition: water, propyl gallate, gallyl glucoside, epigallocatechin gallatyl glucoside) was incorporated in a vehicle formula (INCI composition: aqua, octyldodecyl neopentanoate, octyldodecyl, octyldodecanol, myristyl myristate, acrylates/C10-30 alkyl acrylate crosspolymer, sodium hydroxide, phenoxyethanol, methylparaben, ethylparaben, butylparaben, propylparaben, isobutylparaben). The placebo was made of the formula without the active ingredient. The efficacy of a cosmetic product at 3 % on the reduction of the visibility of dark circles and bags/puffiness under eyes was studied after 15 and 30 days of daily use. The active and placebo were applied twice a day (morning and evening) around the eyes then the area is gently massaged. Evaluation of the reduction of the characteristic colors of the shadows under the eyes: The measurement of the color of the eye circles was done by means of a spectrophotometer/colorimeter CM700d (Konica Minolta). The instrument was able to evaluate the color according to a standard method defined by the International Lighting Commission in 1976 (CIE lab model). The dark color (seen in a dark circle) was represented by contribution of two colors (red and blue). The red color was linked to (a) parameter and the blue color was linked to (b) parameter. The variation of these Relative expression ( % of control) >300 % >200 % and <300 % >30 % and <50 % <30 % Clinical classification dark circles at D15 and D30 no variation 1 slight improvement 2 moderate improvement 3 remarkable improvement 4 Table 2 Clinical scorage for dark circles. Fig. 1 NF- B translocation studies (immuno-fluorescence staining). two parameters ( a and b) was followed by the spectrophotometer. A decrease on the (a) value and an increase of the (b) value indicates the resorption of the black color. Evaluation of the reduction of bags/ puffiness: The measurement of the bags/ puffiness was done by means of a PRIMOS Optical 3D Skin Measuring Device (GFMesstechnik, GmbH). The optical 3D measuring devices PRIMOS was based on digital stripe projection. The technique Classification of the effect strong stimulation stimulation inhibition strong inhibition Table 1 Classification of the effect of the active ingredient. 20 Score allowed to take a high resolution image of the skin, take a 3 dimensional image and analyze by means of image analysis the informations of the 3D image. Clinical evaluation: The dermatologist evaluated the visibility of eye dark circles in accordance with the clinical scores reported in Table 2. Digital Photographs: Digital photographs were taken before and after product use by means of a professional digital reflex camera (Nikon D300, Nikon corporate, Japan) equipped with a macro lens (AF-S Micro Nikkor 60 mm f/2.8 G ED, Nikon corporate, Japan) and a flash system (kit R1C1, Nikon corporate, Japan). Statistical analysis: The inter-group comparisons were performed by Student’s ttest (for paired data). The significance was judged as followed: ns (not significant) p >0.05, *p <0.05; **p <0.01 and ***p< 0.001. SOFW-Journal | 140 | 4-2014 CO SMET ICS I NF LA MMAT I O N ■ Results tent inhibitory effect at the second and highest concentrations (95 and 99 % of inhibition, respectively). Anti-inflammatory studies Effect on NF- B translocation in NHEK (Fig.1): Treatment of NHEK by TNF+IL-1 (stimulated control) induced the nuclear translocation of NF- B from cytosolic to nuclear compartment. The reference, i.e. the association of NF- B inhibitor III + NF- B inhibitor V, inhibited strongly the TNF- +IL-1 -induced NF- B nuclear translocation. The active ingredient at all tested concentrations, did not modify TNF- +IL-1 induced NF- B nuclear translocation. Effect on NF- B activation (Fig. 2 and Table 3): Treatment with increasing concentrations of active ingredient inhibited up to -85 % of the NF- B activity in human cells induced by TNF- . At this stage, we have demonstrated that the active ingredient interacts with NF- B pathway, by inhibiting the NFB activity and not by inhibiting NF- B nuclear translocation. Effect on IL-8 release by NHEK (Fig. 3 and Table 4): In non-stimulated basal conditions, no IL-8 release was detected in NHEK cultures. The activation of NHEK by the association of TNF- + IL-1 resulted in significant IL-8 release (~ 1735 pg/ml). The references NF- B inhibitor III and dexamethasone both importantly inhibited the TNF+ IL-1 - induced IL-8 release by NHEK (66 and 59 % of inhibition, respectively). The active ingredient, tested at 0.016 %, 0.08 % and 0.4 %, presented a dose- dependent inhibitory effect by presenting a moderate inhibitory effect on IL-8 release by activated NHEK at the lowest concentration (55 % of inhibition) and a very strong and po- Effect on CXCL1 release by NHEK (Fig. 4 and Table 5): In non-stimulated basal conditions, CXCL1 release by NHEK was Fig. 2 Regulation of NF- B activity by the active ingredient. Fig. 3 IL-8 release by NHEK. Criteria Concentration in active ingredient ( %) detected (~ 564 pg/ml). The activation of NHEK by the association of TNF+ IL-1 resulted in significant increase of CXCL1 release (~ 1689 pg/ml). In presence of the reference NF- B inhibitor III, CXCL1 release was equivalent to the CXCL1 release observed in TNF(20 ng/mL) Active ingredient + TFN- (20 ng/mL) 4.36 2.18 1.09 0.55 0.27 0.14 0.07 0.03 0.02 0.01 0.04±0.001 0.042±0.001 0.05±0.001 0.06±0.005 0.085±0.01 0.104±0.008 0.186±0.030 0.240±0.035 0.203±0.029 0.246±0.033 Variation in absorbance (VS TNF- alone 0.29 0.29 0.28 0.27 0.24 0.22 0.14 0.09 0.12 0.08 % of inhibition of NFKB pathway 87.66 87.17 84.75 81.75 73.86 68.01 43.09 26.44 37.78 24.69 Absorbance at 650 nm (mean±sem) 0.327±0.040 Table 3 Assay of NF- B activity (Absorbance measurement). SOFW-Journal | 140 | 4-2014 21 CO S MET IC S IN F L A MMAT IO N basal conditions. Thus NF- B inhibitor III almost totally inhibited the TNF- + IL-1 -induced CXCL1 release (99 % of inhibition). The reference dexamethasone induced a significant but only a moderate inhibitory effect on CXCL1 release by activated NHEK (37 % of inhibition). The active ingredient at the lowest test concentration, significantly but moderately inhibited CXCL1 release by activated NHEK. At the second and highest test concentrations, this compound presented a very potent inhibitory effect (134 and 136 % of inhibition, respectively), since the CXCL1 release was below the basal release level. Lymphatic and vascular marker studies Effect on VEGFR-3 and on Heme Oxygenase 1 (HMOX-1) mRNA expressions (Fig. 5): The active ingredient tested at 0.04 % and 0.2 % stimulated strongly the transcription of the VEGFR-3 gene. The relative expressions were respectively 540 and 775 % in comparison to the mean of the three housekeeping genes used. For the gene HMOX-1, only the concentration at 0.2 % induced a significant stimulation effect (294 %). The active ingredient tested at 0.2 % stimulated strongly the transcription of the VEGFR-3 and HMOX-1 genes. Clinical study Clinical effect on dark circles: The use of the product containing active ingredient reduced significantly the dark colour of the dark circles under the eyes as shown by the decrease of a parameter and increase of b parameter (Fig. 6 and Table 6) at D15 and D30. The effect of active ingredient on the monitored parameters was greater than those recorded for placebo. The difference between two Fig. 4 CXCL-1 release by NHEK. Treatment Test compound Stimulated control (IL-1 +TNF- ) Basic data Normalized data Concentration IL-8 (pg/ml) Mean IL-8 (pg/ml) sem (pg/ml) % Stimulated control sem ( %) 5 ng/ml 1602.9 1224.8 2376.9 1734.9 339.1 100 20 <31.3 <31.3 0.0 <2 0 Non-stimulated control p Inhibition ( %) sem ( %) p 0 20 ** 100 0 ** NF- B inhibitor III 5 µM 510.8 619.4 688.4 606.2 51.7 35 3 * 66 3 * Dexamethasone 10 µM 624.5 663.9 899.5 729.3 85.9 42 5 * 59 5 * 0.016 % 969.9 715.1 705.8 796.9 86.5 46 5 ns 55 5 ns 0.08 % 113.3 117.6 113.1 114.7 1.5 7 0 ** 95 0 ** 0.4 % 34 48.5 44.8 42.4 4.4 2 0 ** 99 0 ** Active ingredient Threshold for statistical significance ns: p>0.05, Not significant; *p: 0.01 to 0.05, Significant; **p:0.001 to 0.01, Very significant Table 4 IL-8 release by NHEK. 22 SOFW-Journal | 140 | 4-2014 CO S MET IC S I N F L A MMAT IO N products was statistically significant at all experimental times. The efficacy of the product was also confirmed by both the clinical evaluation carried out by the dermatologist (Table 7) and the majority of volunteers participating in the study (66.7 % of the volunteers found a moderate to remarkable improvement of their dark circles in comparison to only 41.7 % with placebo). Clinical effect on bags/puffiness of the active ingredient: The use of the active ingredient reduced the bags/puffiness volume at D15 and D30 (Table 8 and Fig. 7). The effect of the active ingredient on the monitored parameters was greater than those recorded for placebo, which did not show any evolution upon time (table 8): -10 % of puffiness reduction with a very high statistical significance (p<0,001). This measured effect was visible on the clinical images taken during the study, on which a clear reduction of the bags (puffiness) under eyes as well as a brightening of the dark color can be seen. One must notice that such a reduction was quite good as it is well known that the reduction of puffiness and dark circles on aged volunteers is more difficult than on young ones due to a reduce biological metabolism. ■ Discussion Fig. 5 Relative gene expression of VEGFR3 and HMOX-1 genes (RT-qPCR analysis) CXCL-1 release by NHEK. Treatment Test compound Stimulated control (IL-1 +TNF- ) The active ingredient showed anti-inflammatory like effects on NHEK by strongly inhibiting the NF- B pathway-driven IL-8 and CXCL1 releases. However, the inhibitory effect of the active ingredient was not mediated by a direct inhibition of NF- B translocation but by the inhibition the NF- B activity. These results are Basic data Normalized data Concentration IL-8 (pg/ml) Mean IL-8 (pg/ml) sem (pg/ml) % Stimulated control sem ( %) 5 ng/ml 1536.4 1674.1 1858.5 1689.6 93.3 100 6 785.0 497.1 410.8 564.3 113.1 33 7 Non-stimulated control p Inhibition ( %) sem ( %) p 0 8 ** 100 10 ** NF- B inhibitor III 5 µM 736.7 514.1 476.9 575.9 81.1 34 5 *** 99 7 *** Dexamethasone 10 µM 1234.3 1126.8 1448.6 1269.9 94.6 75 6 * 37 8 * 0.016 % 1144.3 1189.8 1438.3 1257.5 91.3 74 5 * 38 8 * 0.08 % 177.4 <156.3 204.4 <179.4 13.9 <11 1 *** >134 1 *** 0.4 % <156.3 <156.3 <156.3 156.3 0 <9 0 *** >136 0 *** Active ingredient Threshold for statistical significance ns: p>0.05, Not significant; *p: 0.01 to 0.05, Significant; **p:0.001 to 0.01, Very significant; ***p<0.001 extremely significant Table 5 CXCL1 release by NHEK. 24 SOFW-Journal | 140 | 4-2014 CO S MET IC S IN F L A MMAT IO N Fig. 6 Effect of the active ingredient and placebo on a and b parameters. Fig. 7 Digital photography of puffiness and dark circles at D0 (before treatment) and D30 (after treatment with active ingredient) Treatment Test compound Concentration Placebo Active 3% Ingredient Test compund Concentration Placebo Active 3% Ingredient consistent with data from other authors (7, 13). Inhibition of the NF- B pathway was already described for epigallocatechin-3 gallate (EGCG) (7) and gallic acid derivatives (9, 13). EGCG and gallic acid derivatives are component of the active ingredient. It has been shown in the literature that for EGCG, the inhibition is based on the down-regulation of p-I B , p65, p-p65 (7). For gallic acid derivatives the inhibition was reported to be based on the significant inhibition of the nuclear translocation of p65, (a subunit of nuclear factor- B (NF- B)), on the inhibition of the phosphorylation of IkB, I B kinase and p65, the downregulation of the expression of NF- Bregulated inflammatory cytokines (IL-8, TNF- , IL-1 ). This study has also shown that the active ingredient has a role in vessels functions modulation due to its regulation effect observed on two VEGFR3 and HMOX-1 genes. Indeed, the active ingredient improves the drainage function of lymphatic vessels by increasing the expression of VEGFR3 gene; while controling the vascular tone by increasing the expression of the Heme oxygenase-1 gene. The crucial role of VEGFR3 signalling pathway in lymphatic drainage was revealed by the studies of Mendola et al (14) showing that familial lymphedema are due to mutation in VEGFR3 gene. Hagura et al (15) showed that blockage by antibody of VEGFR3 inhibited the drainage function of the lymphatic system (oedema is persistent) and increase the inflammation. VEGFR3 signalling pathway is fundamental for the mediation of drainage function and in the control of skin inflammation. On the other D0 a (mean±sem) (3.39±0.195) D15 a (mean±sem) (3.12±0.179) D30 a (mean±sem) (3±0.0.185) (3.28±0.176) (2.81±0.162) (2.67±0.0.147) a (mean±sem) (-3.05±0.272) a (mean±sem) (-3.02±0.293) a (mean±sem) (-2.8±0.276) (-2.97±0.200) (-2.62±0.185) (-2.42±0.174) % of variation D15 versus D0 D30 versus D0 (-7.2 %)** (-10 %)** (-14.2 %)*** (-17.7 %)*** D15 versus D0 2.3 % ns D30 versus D0 8.9 % ** 11.5 % *** 18.1 % *** Threshold for statistical significance ns : p> 0.05, Not significant;* p : 0.01 to 0.05, Significant, ** p : 0.001 to 0.01, Very significant, ***p >0.001, strongly significant Table 6 Effect of the active ingredient and placebo on a and b parameters. 26 SOFW-Journal | 140 | 4-2014 CO S MET IC S I N F L A MMAT IO N hand, heme oxygenase-1 (HMOX-1) is an enzyme degrading heme into biliverdin, carbon monoxide (CO), and free iron. HMOX-1 protects endothelial cells from apoptosis, is involved in blood-vessel relaxation regulating vascular tone, and participates in blood-vessel formation by means of angiogenesis and vasculogenesis (16). The regulation of vascular tone is due to the relaxation of vascular smooth muscle cells (17). HMOX-1 has been also recognized to have major immunomodulatory and anti-inflammatory properties (17, 18), which have been demonstrated in HMOX-1 knockout mice and a human case of genetic HMOX-1 deficiency. Thus, the literature data have shown that both VEGFR3 and HMOX-1 genes are involved in anti-inflammatory pathways. Our study demonstrated that the active ingredient is able to induce the expression of the HMOX-1 gene, therefore most probably activating these anti-inflammatory properties. Clinical study performed under the control of a dermatologist on human volunteers suffering of dark circles and bags under eyes have shown the efficacy of the active ingredient after 15 and 30 days of treatment. The improvement of dark circles pigmentation was confirmed by the significant variation of a and b parameters measured: decrease of a parameter by about -14 % to -18 % and increase of b parameter by about +11 % and +18 % respectively after 15 and 30 days of treatment. The reduction of bags volumes due to oedema under eyes by about -8 % and -10 % after 15 and 30 days of treatment has demonstrated the improvement of lymphatic drainage provided by the active ingredient Schematic representation showing the anti-inflammatory action of the active ingredient at different level of NF- B pathway. (Fig. 8) ■ Conclusions Dark circles and bags are reported to be one of the biggest concerns for consumers all over the world and more precisely for Asiatic population (Mintel data), right after wrinkles. Both dark circles and under eyes puffiness are associated with a lower self esteem and are reported to have sometimes social impact, as people believe that dark circles are associated with a bad quality of life. Up to now, the toolbox of formulators was limited to few ingredients (caffeine, esculin) or pigments to address this skin issue. By combining three derivatives of gallic acid (gallic acid glucoside, epigallocatechin Treatment Concentration Test compound D0 D15 D30 Dark circles quotation (mean±sem) Dark circles quotation (mean±sem) Dark circles quotation (mean±sem) 2.6±0.101 1.3±0.088 1.6±0.155 2.6±0.101 1.5±0.102 2±0.161 Placebo Active Ingredient glucoside and propyl gallate), which are stabilized forms of gallic acid, it has been possible to obtain significant improvement of dark circles and puffiness on human volunteers in 15 to 30 days. The suggested mechanism of action for this active ingredient in these experiments was the inhibition of the NF- B mediated inflammation. The inhibition is due to the interaction of the active ingredient with the inflammatory NF- B pathway. Indeed, if the active ingredient was not able to block the nuclear translocation of NF- B nuclear factor, we have shown the strong effect of the active ingredient to decrease the transcriptional activation of inflammatory markers (IL 8, CXCL-1) known to be under the control of NF- B nuclear factor after its nuclear translocation. This anti-inflammatory like activity was also detected through the stimulation of the transcription of two important markers HMOX-1 and VEGFR3 known for their anti-inflammatory properties. The reduction of the volume of the bags is expected to be mediated through the activation of the VEGFR3 factor by the active ingredient. This study has highlighted a new efficient target »the NF- B inflammatory pathway« to treat bags and dark circles under eyes. The active ingredient shows 3% Table 7 Clinical quotation of dark circles at D0, D15 and D30 by dermatologists. Treatment Test compound Concentration Placebo Active Ingredient 3% D0 D15 D30 % of variation volume (mean±sem) in mm3 volume (mean±sem) in mm3 volume (mean±sem) in mm3 D15 versus D0 D30 versus D0 21.7±1.334 20.4±1.308 20±1.344 (-6.2 %)** (-8.1 %)** 22.1±1.503 20.2±1.336 19.8±1.340 (-8.4 %)*** (-10.1)*** Threshold for statistical significance ns : p> 0.05, Not significant;* p : 0.01 to 0.05, Significant, ** p : 0.001 to 0.01, Very significant, ***p <0.001, strongly significant Table 8 Effect of the active ingredient and placebo on the volume of the bags under eyes. 28 SOFW-Journal | 140 | 4-2014 More Facts, Less Illusions www.ifscc2015.com CO S MET IC S IN F L A MMAT IO N a direct action on Nf- B pathway by decreasing the transcription rate of NF- B mediated inflammatory markers such as TNF alpha, IL1, IL8, CXCL1, and by reduc- ing the release of CXCL1 and IL8 in the tissue, which leads to the inhibition of the inflammatory cascade. It also shows an indirect interaction with the NF- B signalling pathway by increasing the transcription rate of HMOX-1 known to block nuclear translocation NF- B factor. Meanwhile, the activation of the transcription rate of VEGFR3 can explain the action on the oedematous bags visible under eyes by the improvement of draining properties of lymphatic vessels, which are under the VEGFR3 control. References Fig. 8 Schematic representation of the inflammatory process and the mechanism of action of the active ingredient in inflammatory condition. 30 (1) Giacomoni, P.U., Advancement in skin aging: the future cosmeceuticals. Clin Dermatol, 2008. 26(4): p. 364-6. (2) Roh, M.R. and K.Y. Chung, Infraorbital dark circles: definition, causes, and treatment options. Dermatol Surg, 2009. 35(8): p. 1163-71. (3) Freitag, F.M. and T.F. Cestari, What causes dark circles under the eyes? J Cosmet Dermatol, 2007. 6(3): p. 211-5. (4) Graziosi, A.C., et al., Cutaneous idiopathic hyperchromia of the orbital region (CIHOR): a histopathological study. Aesthetic Plast Surg, 2013. 37(2): p. 434-8. (5) Jiang, D., et al., Influence of Paeonia lactiflora roots extract on cAMP-phosphodiesterase activity and related anti-inflammatory action. J Ethnopharmacol, 2011. 137(1): p. 914-20. (6) Abdelwahab, S.I., Protective mechanism of gallic acid and its novel derivative against ethanol-induced gastric ulcerogenesis: Involvement of immunomodulation markers, Hsp70 and Bcl-2-associated X protein. Int Immunopharmacol, 2013. 16(2): p. 296305. (7) Liu, Q., et al., EGCG attenuates pro-inflammatory cytokines and chemokines production in LPS-stimulated L02 hepatocyte. Acta Biochim Biophys Sin (Shanghai), 2014. 46(1): p. 31-9. (8) Riegsecker, S., et al., Potential benefits of green tea polyphenol EGCG in the prevention and treatment of vascular inflammation in rheumatoid arthritis. Life Sci, 2013. 93(8): p. 307-12. (9) Hsu, H.C., et al., Propyl gallate inhibits TPAinduced inflammation via the nuclear factorkappaB pathway in human THP-1 monocytes. Exp Ther Med, 2013. 5(3): p. 964-968. SOFW-Journal | 140 | 4-2014 CO SMET ICS I NF LA MMAT I O N (11) Auriol Daniel, N.R., Robe Patrick, Lefevre Fabrice, Patent EP 06290972: Phenolic compounds with cosmetic and therapeutic applications: . 14 06 2006. (12) Wittwer, C.T., et al., Continuous fluorescence monitoring of rapid cycle DNA amplification. Biotechniques, 1997. 22(1): p. 130-1, 134-8. (13) Al-Halabi, R., et al., Gallotannin inhibits NFkB signaling and growth of human colon cancer xenografts. Cancer Biol Ther, 2011. 12(1): p. 59-68. (14) Mendola, A., et al., Mutations in the VEGFR3 signaling pathway explain 36 % of familial lymphedema. Mol Syndromol, 2013. 4(6): p. 257-66. (15) Hagura, A., et al., The VEGF-C/VEGFR3 signaling pathway contributes to resolving chronic skin inflammation by activating lymphatic vessel function. J Dermatol Sci, 2013. Abbreviations: NHEK (Normal Human Epidermal Keratinocytes), NF- B (Nuclear Factor-kappa B), TNF- (Tumor Necrosis Factor alpha), IL1- (Interleukin-1 alpha), IL8 (Interleukin-8), CXCL1 (Chemokine C-X-C motif ligand 1), VEGFR3 (Vascular endothelial growth factor receptor 3), HMOX-1 (Heme Oxygenase -1). (16) Loboda, A., et al., Heme oxygenase-1 and the vascular bed: from molecular mechanisms to therapeutic opportunities. Antioxid Redox Signal, 2008. 10(10): p. 1767-812. (17) Immenschuh, S. and H. Schroder, Heme oxygenase-1 and cardiovascular disease. Histol Histopathol, 2006. 21(6): p. 679-85. (18) Paine, A., et al., Signaling to heme oxygenase-1 and its anti-inflammatory therapeutic potential. Biochem Pharmacol, 2010. 80(12): p. 1895-903. *Authors´ address: Dr. Hanane Chajra Dr. Daniel Auriol Dr. Kuno Schweikert Dr. Fabrice Lefevre Induchem AG Industriestr. 8 8604 Volketswil Switzerland www.induchem.com ■ ADD A TOUCH OF GENIUS TO YOUR HAIR CARE FORMULATIONS! it u s ! a n d v is Come cs ti e m s in -c o rg Hambu 4 r il 2 0 1 1– 3 Ap 0 B o o th 1K2 With Baycusan® C 1008 from Bayer, you can now easily incorporate a three-way advantage into your future hair care products. Discover the amazing possibilities of this innovative polymer! Protection against heat, humidity and damage Repair of split ends, proven frizz control Styling: instant and lasting natural strong hold Curious? Visit www.bayercosmetics.com or contact us at email@example.com SOFW-Journal | 140 | 4-2014 P O LY U R E T H A N E S F O R C O S M E T I C S 31 Bayer MaterialScience AG, 51368 Leverkusen, Germany · MS00067300 (10) Massaro, F.C., et al., Cerumen of Australian stingless bees (Tetragonula carbonaria): gas chromatography-mass spectrometry fingerprints and potential anti-inflammatory properties. Naturwissenschaften, 2011. 98(4): p. 329-37.