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Background Monitoring the transcutaneous permeation of exogenous molecules using conventional techniques generally requires long pre-analytical preparation or labelling of samples. However, Raman spectroscopy is a label-free and non-destructive method which provides spatial distribution of tracked actives in skin. The aim of our study was to prove the interest of Raman imaging coupled with multivariate curve resolution alternating least square (MCR-ALS) analysis in monitoring retinol penetration into frozen and living human skin. Materials and methods After topical treatment of skin samples by free or encapsulated retinol, thin cross sections were analysed by Raman imaging (up to 100 µm depth). Mann-Whitney test was used to identify retinol spectroscopic markers in skin. MCR-ALS was used to estimate retinol contribution in Raman spectral images. Heat maps were constructed to compare the distribution of free and encapsulated retinol in skin models. Results We identified the bands at 1158, 1196 and 1591 cm-1 as specific features for monitoring retinol in skin. Moreover, our MCR-ALS results showed an improvement of retinol penetration (up to 30 µm depth) with the encapsulated form as well as storage reservoir formation in stratum corneum, for each skin model. Finally, greater retinol penetration into living skin was observed. Conclusion This study shows a proof of concept for the evaluation of retinol penetration in skin using Raman imaging coupled with MCR-ALS. This concept needs to be validated on more subjects to include inter-individual variability but also other factors affecting skin permeation (age, sex, pH, etc). Our study can be extended to other actives.

(1) Background: Preclinical studies report that the ethanolic fraction from

Skin lipids are essential in every compartment of the skin where they play a key role in various biological functions. Interestingly, their role is central in the maintenance of hydration which is related to skin barrier function and in the skin structure through adipose tissue. It is well described today that skin lipids are affected by ageing giving skin sagging, wrinkles and dryness. Thereby, developing cosmetic actives able to reactivate skin lipids would be an efficient ant-ageing strategy. Due to the strong commitment of our scientists to innovate responsibly and create value, they designed a high value active ingredient named here as Vetiver extract, using a ground-breaking upcycling approach. We evidenced that this unique extract was able to reactivate globally the skin lipids production, bringing skin hydration and plumping effect for mature skin.In order to demonstrate the global renewal of lipids, we evaluated the lipids synthesis on cutaneous cells that produce lipids such as keratinocytes, sebocytes and adipocytes then on Reconstructed Human Epidermis and skin explants. We evaluated the expression of proteins involved in ceramides transport and barrier cornification. We then evaluated hydration and sebaceous parameters on a panel of mature volunteers.We firstly demonstrated that Vetiver extract induced sebum production from human sebocytes cells lines but also improved its quality as observed by the production of specific antimicrobial lipids. Secondly, we demonstrated that Vetiver extract was able to restore skin barrier with the increase of skin lipids neosynthesis on Reconstructed Human Epidermis and skin explants. We also evidenced that Vetiver extract stimulated the lipids transport and epidermal cornification. Finally, Vetiver extract showed a significant effect on adipogenesis and maturation of adipocytes at in vitro and ex vivo models. We confirmed all these activities by showing that Vetiver extract improved sebum production and brought hydration through an increase of lipids content and their conformation. Vetiver extract induced an improvement of skin fatigue and a plumping effect by acting deeply on adipose tissue.In conclusion, we developed an active ingredient able to bring anti-ageing effect for mature skin by a global increase of skin lipids.Les lipides de la peau sont essentiels dans chaque compartiment de la peau où ils jouent un rôle clé dans diverses fonctions biologiques. Il est intéressant de noter que leur rôle est central dans le maintien de l'hydratation, liée à la fonction de barrière cutanée, mais aussi dans la structure même de la peau, par le biais du tissu adipeux. Il est bien décrit aujourd'hui que les lipides de la peau sont affectés par le vieillissement, ce qui entraîne un relâchement de la peau, des rides et une sécheresse. Ainsi, le développement d'actifs cosmétiques capables de réactiver les lipides de la peau serait une stratégie efficace de lutte contre le vieillissement. En raison de l'engagement fort de nos scientifiques à innover de manière responsable et à créer de la valeur, ils ont conçus un ingrédient actif à forte valeur ajoutée, appelé ici extrait de Vétiver, en utilisant une approche révolutionnaire de « up-cycling ». Nous avons démontré que cet extrait unique était capable de réactiver globalement la production de lipides de la peau, apportant une hydratation de la peau et un effet repulpant pour les peaux matures. MÉTHODES: Afin de démontrer le renouvellement global des lipides, nous avons évalué la synthèse des lipides sur les cellules cutanées qui produisent des lipides tels que les kératinocytes, les sébocytes et les adipocytes, puis sur un modèle d’Epiderme Humain Reconstruit et les explants de peau. Nous avons évalué l'expression des protéines impliquées dans le transport des céramides et la kératinisation de la barrière cutanée. Nous avons ensuite évalué l'hydratation et les paramètres sébacés sur un panel de volontaires matures. RÉSULTATS: Nous avons tout d'abord démontré que l'extrait de Vétiver induit la production de sébum à partir de lignées cellulaires de sébocytes humains mais améliore également sa qualité comme l'indique la production de lipides antimicrobiens spécifiques. Ensuite, nous avons démontré que l'extrait de Vétiver était capable de restaurer la barrière cutanée grâce à l'augmentation de la néosynthèse lipidique sur un modèle d’Epiderme Humain Reconstruit et sur des explants de peau. Nous avons également démontré que l'extrait de Vétiver stimulait le transport des lipides et la kératinisation de l'épiderme. Enfin, l'extrait de Vétiver a montré un effet significatif sur l'adipogenèse et la maturation des adipocytes dans des modèles in vitro et ex vivo. Nous avons confirmé à l’échelle clinique toutes ces activités en montrant que l'extrait de Vétiver améliorait la production de sébum et apportait une hydratation grâce à une augmentation de la teneur en lipides ainsi qu’une modification de leur conformation. L'extrait de Vétiver a induit une amélioration de la fatigue cutanée et un effet repulpant en agissant en profondeur sur le tissu adipeux.En conclusion, nous avons développé un ingrédient actif capable d'apporter un effet anti-âge aux peaux matures par une augmentation globale des lipides de la peau.

Background Intrinsic aging promotes wrinkles formation by an imbalance between matrix synthesis/degradation in favor of degradation. This is accelerated by the exposome leading to overproduction of protease and fewer remodeling. Objective Protecting the integrity of extracellular matrix appears as the most efficient anti-aging solution. We developed a grafted HA specifically designed to get anti-aging property due to a specific molecular weight and acetylation degree. Methods A transcriptomic analysis was performed on fibroblasts, followed by a measurement of MMP secretion and subsequent effect on collagen degradation. MMP expression in skin explants concerned by chronobiological and extrinsic aging was analyzed by immunostaining. A clinical study was conducted on volunteers presenting wrinkles on face to evaluate flash reduction of wrinkles after 6 h of application by profilometry and anti-aging efficacy after 2 months by VISIA® CR2.3. Results Transcriptomic analysis evidenced an inhibition of MMP gene expression with acetylated HA, confirmed by an inhibition of MMPs release by fibroblasts, and a protection of type I collagen against degradation. We confirmed the reduction of MMPs in mature skin and in skin explants exposed to UV and urban dust. We demonstrated during clinical studies the flash reduction effect of acetylated HA on crow's feet wrinkles and a filling of nasogenian areas 6 h after application, and a wrinkles number reduction on nasogenian area up to 2 months of application. Conclusion We developed a new grafted HA owing protective properties against ECM degradation induced by chronobiological and extrinsic aging, leading to a significant and efficient anti-wrinkles effect.

Background Several studies evidenced significant increase of cortisol is the consequence of UV or emotional stress and leads to various deleterious effects in the skin. Aim The well-aging, a new concept of lifestyle, procures an alternative to the anti-aging strategy. We demonstrated that Tephrosia purpurea extract is able to stimulate well-being hormones while reducing cortisol release. Furthermore, we hypothesized that the extract could positively influence the global skin homeostasis. Method We evaluated the impact of the extract on cortisol, β-endorphin, and dopamine, released by normal human epidermal keratinocytes (NHEKs). A gene expression study was realized on NHEKs and NHDFs. The protein over-expression of HMOX1 and NQO1 was evidenced at cellular and tissue level. Finally, we conducted a clinical study on 21 women living in a polluted environment in order to observe the impact of the active on global skin improvement. Results The extract is able to reduce significantly the cortisol release while inducing the production of β-endorphin and dopamine. The gene expression study revealed that Tephrosia purpurea extract up-regulated the genes involved in antioxidant response and skin renewal. Moreover, the induction of HMOX and NQO1 expression was confirmed on NHDFs, NHEKs and in RHE. We clinically demonstrated that the extract improved significantly the skin by reducing dark circles, represented by an improvement of L*, a*, and ITA parameters. Conclusion Tephrosia purpurea extract has beneficial effects on skin homeostasis through control of the well-being state and antioxidant defenses leading to an improvement of dark circles, a clinical features particularly impacted by emotional and environmental stress.

Background Healthy skin is a delicate balance between skin renewal and microbiota homeostasis, and its imbalance promotes premature aging and dermatological disorders. Skin stem cells are key actors in this process but their sensitivity to aging and external stressors such as UV reduces the skin renewal power. The skin microbiota has been recently described as active in the healthy skin, and its imbalance could trigger some disorders. Aims We hypothesized that reactivation of stem cells and maintenance of microbiota could be a disruptive strategy for younger and healthier skin. We thus developed a new plant extract that restores the entire skin renewal process by sequential activation from stem cells stimulation to microbiota protection. Methods We studied stem cells comportment in the presence of Orobanche rapum extract by survivin immunocytochemistry and caspases 3 and 9 dosages. We also analyzed epidermal differentiation markers by immunohistochemistry and lipids organization by GC/MS At the clinical level, we investigated the impact of O. rapum extract on microbiota and on skin aspect. Results We demonstrated an active protection of skin stem cells through the maintenance of their clone-forming capacity and resistance to UV through the overexpression of survivin coupled to caspases inhibition. Furthermore, we showed the restoration of epidermal differentiation markers and ceramide biosynthesis favorable to orthorhombic organization. Clinical studies, including microbiota analysis, showed an active skin surface renewal coupled with microbiota protection. Conclusion We evidenced that our active ingredient is able to stimulate skin rejuvenation while protecting the cutaneous microbiota, creating healthier skin and thereby beauty.

Objective In hair care cosmetic products’ evaluation, one commonly used method is to evaluate the hair appearance as a gold standard in order to determine the effect of an active ingredient on the final state of the hair via visual appreciation. Although other techniques have been proposed for a direct analysis of the hair fibres, they give only surface or structural information, without any accurate molecular information. A different approach based on confocal Raman spectroscopy has been proposed for tracking in situ the molecular change in the keratin directly in the human hair fibres. It presents a high molecular specificity to detect chemical interactions between molecules and can provide molecular information at various depths at the cortex and cuticle levels. Methods To evaluate the potential of confocal Raman spectroscopy in testing the efficiency of cosmetic ingredients on keratin structure, we undertook a pilot study on the effectiveness of a smoothing shampoo on natural human hair, by analysing α‐helix and β‐sheet spectral markers in the Amide I band and spectral markers specific to the cystin sulfur content. Results We confirmed that an active proved to be effective on a gold standard decreases α‐helix keratin conformation and promotes β‐sheet keratin conformation in the hair fibres. We also showed that treatment with the effective active decreases the intensity of covalent disulfide (S–S at 510 cm‐1) cross‐linking bands of cysteine. These data confirm that the effective active also acts on the tertiary structure of keratin. Conclusion From these experiments, we concluded that the effective active has a smoothing effect on the human hair fibres by acting on α‐helix and β‐sheet keratin conformation and on the tertiary structure of keratin. Based on these results, confocal Raman spectroscopy can be considered a powerful technique for investigating the influence of hair cosmetic ingredients on keratin structure in human hair fibres. Moreover, this analytical technique has the advantage of being non‐destructive and label free; in addition, it does not require sample extraction or purification and it can be applied routinely in cosmetic laboratories., Confocal Raman spectroscopy can be considered as a powerful technique for investigating the influence of hair cosmetic ingredients on keratin structure in human hair fibres.

International audience; The aim of the present study was to investigate the neuro-soothing activity of a water-soluble hydrolysate obtained from the red microalgae Rhodosorus marinus Geitler (Stylonemataceae). Transcriptomic analysis performed on ≈100 genes related to skin biological functions firstly revealed that the crude Rhodosorus marinus extract was able to significantly negatively modulate specific genes involved in pro-inflammation (interleukin 1α encoding gene, IL1A) and pain detection related to tissue inflammation (nerve growth factor NGF and its receptor NGFR). An in vitro model of normal human keratinocytes was then used to evaluate the ability of the Rhodosorus marinus extract to control the release of neuro-inflammation mediators under phorbol myristate acetate (PMA)-induced inflammatory conditions. The extract incorporated at 1% and 3% significantly inhibited the release of IL-1α and NGF secretion. These results were confirmed in a co-culture system of reconstructed human epithelium and normal human epidermal keratinocytes on which a cream formulated with the Rhodosorus marinus extract at 1% and 3% was topically applied after systemic induction of neuro-inflammation. Finally, an in vitro model of normal human astrocytes was developed for the evaluation of transient receptor potential vanilloid 1 (TRPV1) receptor modulation, mimicking pain sensing related to neuro-inflammation as observed in sensitive skins. Treatment with the Rhodosorus marinus extract at 1% and 3% significantly decreased PMA-mediated TRPV1 over-expression. In parallel with these biological experiments, the crude Rhodosorus marinus extract was fractionated by centrifugal partition chromatography (CPC) and chemically profiled by a recently developed 13C NMR-based dereplication method. The CPC-generated fractions as well as pure metabolites were tested again in vitro in an attempt to identify the biologically active constituents involved in the neuro-soothing activity of the Rhodosorus marinus extract. Two active molecules, namely, γ-aminobutyric acid (GABA) and its structural derivative GABA-alanine, demonstrated a strong capacity to positively regulate skin sensitization mechanisms related to the TRPV1 receptors under PMA-induced inflammatory conditions, therefore providing interesting perspectives for the treatment of sensitive skins, atopia, dermatitis, or psoriasis.

International audience; Barks from conifers and broadleaved trees constitute abundant wastes generated from wood harvesting and logging activities. Extracts of such residues obtained from Alnus trees have been reported as interesting resources with potent antibacterial activities. The present study aims to determine the antimicrobial activity of a crude methanol extract prepared from the bark of Alnus glutinosa against a panel of 22 bacteria and yeasts and to optimize a purification method enabling the high production of the most active substances. Fractionation of the crude extract was performed by Centrifugal Partition Chromatography (CPC) using a three-phase solvent system composed of n-heptane, methyl-ter-butyl ether, acetonitrile and water. The major known compounds contained in the fractions produced by CPC were chemically profiled by 13C NMR dereplication, resulting in the unambiguous identification of oregonin, hirsutanonol, betulinic acid, and alusenone 1a. The antibacterial evaluation of the fractions by bioautography on Staphylococcus aureus revealed that oregonin, in addition to being the major metabolite of the crude extract (∼32% w/w), was the most active with an antibacterial inhibitory effect comparable to antibiotics. The purification of oregonin was optimized at the laboratory-scale by CPC. A single injection of 3.7 g of crude extract resulted in a recovery of 72% (850 mg) of the available oregonin at purity higher than 94%.

Background: Topical delivery of molecules into the human skin is one of the main issues in dermatology and cosmetology. Several techniques were developed to study molecules penetration into the human skin. Although widely accepted, the conventional methods such as Franz diffusion cells are unable to provide the accurate localization of actives in the skin layers. A different approach based on Raman spectroscopy has been proposed to follow-up the permeation of actives. It presents a high molecular specificity to distinguish exogenous molecules from skin constituents. Methods: Raman micro-imaging was applied to monitor the skin penetration of hyaluronic acids (HA) of different molecular weights. The first step, was the spectral characterization of these HA. After, we have determined spectral features of HA by which they can be detected in the skin. In the second part, transverse skin sections were realized and spectral images were recorded. Results: Our results show a difference of skin permeation of the three HA. Indeed, HA with low molecular weight (20– 300 kDa) passes through the stratum corneum in contrast of the impermeability of high molecular weight HA (1000– 1400 kDa). Conclusion: Raman spectroscopy represents an analytical, non-destructive, and dynamic method to evaluate the permeation of actives in the skin layers.