In vitro, ex vivo and clinical approaches to evaluate the potential effect of Gentiana lutea extract on skin

International audienceAbstract Background Dark circles affect subjects of all ages and in all skin types. They can be treated by various methods, particular by topical solutions. This investigation was directed towards exploring the effect of gentiopicroside (GP) on the skin around the eyes. For this, an extract of Gentiana lutea (GIE) containing GP (65% by dry matter) was evaluated on oxidant and angiogenesis parameters using in vitro and ex‐vivo studies. A clinical experimentation was also realized. Methods The effect of GIE at different concentrations on antioxidant gene was evaluated in vitro by RT‐qPCR after treatment of NHDF. The effect of 2.93 μg mL −1 GIE on the release of VEGF‐A and VEGF‐C by NHDF was also studied. The effect of 87.9 μg mL −1 GIE was also evaluated on pseudotube formation in a coculture system of normal dermal microvascular endothelial cells (HMVEC‐d)‐NHDF stimulated or not with VEGF as pro‐angiogenic factor. Prior to these assays, preliminary cytotoxicity assays were performed using a standard WST‐8 reduction assay. The expressions of carboxymethyl‐lysine and glyoxalase‐1 were quantified on skin explants topically treated with 147 μg mL −1 GIE in basal and UVA‐irradiated conditions. A clinical study was conducted in 22 subjects using topical twice daily for 14 days on eye area (split‐face application: cream containing 147 μg mL −1 GIE versus placebo). 3D image acquisition and skin colour measurement were performed at D0 and D14. Results Treatment of GIE upregulated the gene expression of NFE2L2 and downregulated the expression of CXCL8. GIE targeted AGEs pathways and reduced the formation of pseudotubes. A total of 147 μg mL −1 GIE gel cream significantly reduced significantly the average roughness and relief of the upper eyelid skin as well as the redness of dark circles after 14 days of application. Conclusion By acting on the pathway of AGEs, VEGF‐A and VEFG‐C, GIE seems to allow a rejuvenation of the skin resulting, among others, in a decrease in redness. It now would be interesting to evaluate the efficacy of GIE on skin around eyes microbiota, antibacterial gentiopicroside property being well‐established.Résumé Contexte Le contour des yeux est une zone sensible. Les cernes affectent les sujets de tout âge et de tout type de peau. Différentes solutions peuvent être proposées, dont les solutions topiques. Cette étude visait a explorer l'effet d’un extrait de Gentiana lutea (GIE) riche en gentiopicroside (65% de matière sèche) sur des paramètres d'oxydation et d'angiogenèse au moyen d’études in vitro et ex‐vivo. Une expérimentation clinique a également été réalisée. Méthodes L'effet du GIE a différentes concentrations sur des gènes antioxydants a été évalué in vitro par RT‐qPCR après traitement de fibroblastes (NHDF). L'effet de 2.93 μg mL−1 GIE sur la libération de VEGF‐A et VEGF‐C a également été étudié. Il en est de même pour l'effet de 87.9 μg g mL−1 GIE sur la formation de pseudotubes qui a été évalué dans un système de co‐culture de cellules endothéliales (HMVEC‐d)‐ NHDF stimulées ou non avec du VEGF comme facteur pro‐angiogénique. Les expressions de la carboxymethyl‐lysine et de la glyoxalase‐1 ont été quantifiées sur des explants cutanés traites par voie topique avec 147 μg g mL−1 GIE dans des conditions basales et irradiées par UVA. Une étude clinique a été menée sur vingt‐deux sujets en utilisant un traitement topique deux fois par jour pendant 14 jours sur le contour des yeux (crème contenant 147 μg g mL−1 GIE contre placebo). L'acquisition d'images 3D et la mesure de la couleur de la peau ont été réalisées a J0 et J14. Résultats Le traitement par GIE a augmenté l'expression génétique de NFE2L2 et diminue l'expression de CXCL8. GIE a cible les voies des AGEs et a réduit la formation de pseudotubes. 147 μg g mL−1 GIE gel crème a significativement réduit la rugosité moyenne et le relief de la peau de la paupière supérieure ainsi que la rougeur des cernes après 14 jours d'application. Conclusion En agissant sur la voie des AGEs, du VEGF‐A et du VEFG‐C, GIE semble permettre un rajeunissement de la peau se traduisant, entre autres, par une diminution des rougeurs. Il serait maintenant intéressant d'évaluer l'efficacité du GIE sur le microbiote de la peau du contour des yeux, la propriété antibactérienne du gentiopicroside étant bien établie

Development of polymorphic microsatellite markers issued from pyrosequencing technology for the medicinal mushroom Agaricus subrufescens

International audienceThe recently described procedure of microsatellite-enriched library pyrosequencing was used to isolate microsatellite loci in the gourmet and medicinal mushroom Agaricus subrufescens. Three hundred and five candidate loci containing at least one simple sequence repeats (SSR) locus and for which primers design was successful, were obtained. From a subset of 95 loci, 35 operational and polymorphic SSR markers were developed and characterized on a sample of 14 A.similar to subrufescens genotypes from diverse origins. These SubSSR markers each displayed from two to 10 alleles with an average of 4.66 alleles per locus. The observed heterozygosity ranged from 0 to 0.71. Several multiplex combinations can be set up, making it possible to genotype up to six markers easily and simultaneously. Cross-amplification in some closely congeneric species was successful for a subset of loci. The 35 microsatellite markers developed here provide a highly valuable molecular tool to study genetic diversity and reproductive biology of A.similar to subrufescens

Rhodomyrtus tomentosa Fruit Extract and Skin Microbiota: A Focus on C. acnes Phylotypes in Acne Subjects

International audienceKnowing that Rhodomyrtus tomentosa is known to have antibacterial effects, this study investigated the skin microbiota with a focus on Cutibacterium acnes (C. acnes) phylotypes in subjects with acne, and determined microbiota changes after 28 days of treatment with berries Rhodomyrtus tomentosa as an active ingredient (RT). Skin swabs from seventeen acne subjects were collected and the skin microbiome was analyzed using 16S rRNA gene sequencing. A culture-independent next-generation sequencing (NGS)-based SLST (single-locus sequence typing) approach was aimed at evaluating RT extract effects on C. acnes phylotype repartition. Clinical evaluations (lesion counts) were performed at baseline (D0) and after 28 days (D28) of twice-daily application of the RT active ingredient. We determined: (1) the skin microbiota at D0 was dominated by Actinobacteria followed by Firmicutes and Proteobacteria; (2) at the genus level, Cutibacterium was the most abundant genus followed by Staphylococcus and Corynebacterium; (3) C. acnes was the major species in terms of mean abundance, followed by Staphylococcus epidermidis (S. epidermidis) and Staphylococcus hominis (S. hominis); and (4) phylotype IA1 was most represented, with a predominance of SLST type A1, followed by phylotypes II, IB, IA2, IC, and III. After 28 days of RT extract treatment, phylotype repartition were modified with a decrease in abundance (approximately 4%) of phylotype IA1 and an increase in phylotype II and III. Cutibacterium granulosum (C. granulosum) abundance also decreased. Reduction of retentional and inflammatory lesions was also noted only after RT treatment; thus, RT extract acts as a microbiota-regulating agent

The genetic linkage map of the medicinal mushroom Agaricus subrufescens reveals highly conserved macrosynteny with the congeneric species Agaricus bisporus

Comparative linkage mapping can rapidly facilitate the transfer of genetic information from model species to orphan species. This macrosynteny analysis approach has been extensively used in plant species, but few example are available in fungi, and even fewer in mushroom crop species. Among the latter, the Agaricus genus comprises the most cultivable or potentially cultivable species. Agaricus bisporus, the button mushroom, is the model for edible and cultivable mushrooms. We have developed the first genetic linkage map for the basidiomycete A. subrufescens, an emerging mushroom crop known for its therapeutic properties and potential medicinal applications. The map includes 202 markers distributed over 16 linkage groups (LG), and covers a total length of 1701 cM, with an average marker spacing of 8.2 cM. Using 96 homologous loci, we also demonstrated the high level of macrosynteny with the genome of A. bisporus. The 13 main LG of A. subrufescens were syntenic to the 13 A. bisporus chromosomes. A disrupted synteny was observed for the three remaining A. subrufescens LG. Electronic mapping of a collection of A. subrufescens expressed sequence tags on A. bisporus genome showed that the homologous loci were evenly spread, with the exception of a few local hot or cold spots of homology. Our results were discussed in the light of Agaricus species evolution process. The map provides a framework for future genetic or genomic studies of the medicinal mushroom A. subrufescens