Strat-M® positioning for skin permeation studies: A comparative study including EpiSkin® RHE, and human skin.

In the development and optimization of dermatological products, In Vitro Permeation Testing (IVPT) is pivotal for controlled study of skin penetration. To enhance standardization and replicate human skin properties reconstructed human skin and synthetic membranes are explored as alternatives. Strat-M® is a membrane designed to mimic the multi-layered structure of human skin for IVPT. For instance, in Strat-M®, the steady-state fluxes (JSS) of resorcinol in formulations free of permeation enhancers were found to be 41 ± 5 µg/cm2·h for the aqueous solution, 42 ± 6 µg/cm2·h for the hydrogel, and 40 ± 6 µg/cm2·h for the oil-in-water emulsion. These results were closer to excised human skin (5 ± 3, 9 ± 2, 13 ± 6 µg/cm2·h) and surpassed the performance of EpiSkin® RHE (138 ± 5, 142 ± 6, and 162 ± 11 µg/cm2·h). While mass spectrometry and Raman microscopy demonstrated the qualitative molecular similarity of EpiSkin® RHE to human skin, it was the porous and hydrophobic polymer nature of Strat-M® that more faithfully reproduced the skin's diffusion-limiting barrier. Further validation through similarity factor analysis (∼80-85%) underscored Strat-M®'s significance as a reliable substitute for human skin, offering a promising approach to enhance realism and reproducibility in dermatological product development.

Skin and eye irritancy assessment of six lactic acid bacteria strains.

Here we investigate the suitability of in vitro models to assess the skin and eye irritation potential of six microbial strains. Acute skin irritation was tested according to the unmodified and modified OECD test guideline (OECD TG) 439, while acute eye irritation was examined using the OECD TG 491 and 492. The OECD TG 439 guideline, modified to introduce 8-10 µg/mL of streptomycin during the recovery phase and use of test items containing 100% microbial product instead of finished formulae, was found to be suitable for skin irritation evaluation. On the other hand, the OECD TG 491 procedure was the most appropriate for evaluating eye irritation. None of the six microbial strains, namely, Lactiplantibacillus plantarum (IMI 507026, IMI 507027, IMI 507028), Lacticaseibacillus rhamnosus (IMI 507023), and Pediococcus pentosaceus (IMI 507024, IMI 507025), tested in this study caused skin or eye irritation under the study condition.

Improved Tool for Predicting Skin Irritation on Reconstructed Human Epidermis Models Based on Electrochemical Impedance Spectroscopy.

The rabbit skin irritation test has been the standard for evaluating the irritation potential of chemicals; however, alternative methods that do not use animal testing are actively encouraged. Reconstructed human epidermis (RhE) models mimic the biochemical and physiological properties of the human epidermis and can be used as an alternative method. On RhE methods, the metabolic activity of RhE models is used to predict skin irritation, with a reduction in metabolic activity indicating a reduced number of viable cells and linking cell death to skin irritation processes. However, new challenges have emerged as the use of RhE models increases, including the need for non-invasive and marker-free methodologies to assess cellular states. Electrochemical impedance spectroscopy (EIS) is one such methodology that can meet these requirements. In this study, our results showed that EIS can differentiate between irritant and non-irritant chemicals, with a significant increase in the capacitance values observed in the irritant samples. A ROC curve analysis showed that the prediction method based on EIS met OECD TG 439 requirements at all time points and had 95% within-laboratory reproducibility. Comparison with the MTT viability assay showed that prediction using EIS achieved higher sensitivity, specificity, and accuracy. These results suggest that EIS could potentially replace animal testing in the evaluation of irritation potential and could be a valuable addition to in vitro testing strategies.

Investigating the surfactant antagonism with the Open Source Reconstructed Epidermis (OS-Rep) model.

Under the current EU chemicals legislation, in vitro test methods became the preferred methods to identify and classify the skin irritation potential of chemicals and mixtures. Among these, especially in vitro skin models are widely used. For surfactants, a well-known group of typically irritating chemicals, it is a long-standing experience that the irritation potential of a mixture of surfactants is typically lower than the irritation potential of the single surfactants, an effect usually described as surfactant antagonism. In order to evaluate if this effect can be observed in skin model systems as well, the irritation potential of the surfactants and of their mixtures was determined in the Open Source Reconstructed Epidermis (OS-REp) models. Combinations of sodium dodecyl sulfate or linear alkylbenzene sulfonate with cocoamidopropyl betain and alkyl polyglycosid, respectively, resulted in a clear decrease of the irritation potential compared to the irritation exerted by the single surfactants. The effect appeared to be primarily driven by the mixture's lower ability to damage the skin model's barrier, as shown by a reduced fluorescein permeation.

Validation study for in vitro skin irritation test using reconstructed human skin equivalents constructed by layer-by-layer cell coating technology.

The aim of this study is to validate an in vitro skin irritation test (SIT) using three-dimensional reconstructed human epidermal (RhE) skin equivalents prepared by layer-by-layer (LbL) method (LbL-3D Skin) in a series of interlaboratory studies. The goal of these validation studies is to evaluate the ability of this in vitro test to reliably discriminate skin irritant from nonirritant chemicals, as defined by OECD and UN GHS. This me-too validation study is to assess the within- and between-laboratory reproducibility, as well as the predictive capacity, of the LbL-3D Skin SIT in accordance with performance standards for OECD TG 439. The developed skin model, LbL-3D Skin had a highly differentiated epidermis and dermis, similar to the validated reference methods (VRM) and native human skin. The quality parameters (cell survival in controls, tissue integrity, and barrier function) were similar to VRM and in accordance with OECD TG 439. The LbL-3D Skin SIT validation study was performed by three participating laboratories and consisted of three independent tests using 20 reference chemicals. The results obtained with the LbL-3D Skin demonstrated high within-laboratory and between-laboratory reproducibility, as well as high accuracy for use as a stand-alone assay to distinguish skin irritants from nonirritants. The predictive potency of LbL-3D Skin SIT using total 54 test chemicals were comparable to those in other RhE models in OECD TG 439. The validation study demonstrated that LbL-3D Skin has proven to be a robust and reliable method for predicting skin irritation.

Uso del dermatoscopio en la lectura de pruebas epicutáneas: ¿qué puede aportar? / Dermoscopy in Patch Testing: How Can It Help?

La lectura de los parches de pruebas epicutáneas no es sencilla, presenta una gran variabilidad inter- e intraobservador y requiere experiencia. En ocasiones es realmente difícil determinar si se trata de una reacción alérgica de intensidad leve o si estamos ante una reacción irritativa. Recientemente se han publicado algunos trabajos que han estudiado las características dermatoscópicas de las distintas reacciones que se producen tras realizar pruebas epicutáneas. La característica dermatoscópica más frecuentemente observada en los parches alérgicos es el eritema homogéneo, si bien vasos puntiformes, vesículas o costras y áreas amarillo-anaranjadas también parecen indicar una reacción alérgica, guardando cierta similitud con lo observado en patología inflamatoria como en el eccema. Por otro lado, en cuanto a las reacciones irritativas, el patrón más indicativo sería el «patrón del poro», acompañado de eritema perifolicular. Estos hallazgos dermatoscópicos pueden ser de utilidad al clínico en su toma de decisiones ante una reacción dudosa (AU)

Interpreting patch test reactions is not easy. It requires experience and is characterized by high intraobserver and interobserver variability. It can sometimes be truly difficult to discern between a weak allergic reaction and an irritant reaction. A number of recent studies have investigated the dermoscopic features of patch test reactions. Homogeneous erythema is the main feature observed in patients with a positive allergic reaction, although dotted vessels, vesicles, crusts and yellow-orange areas may also provide clues. These features are somewhat similar to those observed in inflammatory conditions, such as eczema. In patients with an irritant reaction, the most common dermoscopic findings are the pore reaction pattern and perifollicular erythema. Dermoscopy could be useful for establishing a diagnosis in the case of doubtful patch test reactions (AU)

[Translated article] Dermoscopy in Patch Testing: How Can It Help? / Uso del dermatoscopio en la lectura de pruebas epicutáneas: ¿qué puede aportar?

Interpreting patch test reactions is not easy. It requires experience and is characterized by high intraobserver and interobserver variability. It can sometimes be truly difficult to discern between a weak allergic reaction and an irritant reaction. A number of recent studies have investigated the dermoscopic features of patch test reactions. Homogeneous erythema is the main feature observed in patients with a positive allergic reaction, although dotted vessels, vesicles, crusts and yellow-orange areas may also provide clues. These features are somewhat similar to those observed in inflammatory conditions, such as eczema. In patients with an irritant reaction, the most common dermoscopic findings are the pore reaction pattern and perifollicular erythema. Dermoscopy could be useful for establishing a diagnosis in the case of doubtful patch test reactions (AU)

La lectura de los parches de pruebas epicutáneas no es sencilla, presenta una gran variabilidad inter- e intraobservador y requiere experiencia. En ocasiones es realmente difícil determinar si se trata de una reacción alérgica de intensidad leve o si estamos ante una reacción irritativa. Recientemente se han publicado algunos trabajos que han estudiado las características dermatoscópicas de las distintas reacciones que se producen tras realizar pruebas epicutáneas. La característica dermatoscópica más frecuentemente observada en los parches alérgicos es el eritema homogéneo, si bien vasos puntiformes, vesículas o costras y áreas amarillo-anaranjadas también parecen indicar una reacción alérgica, guardando cierta similitud con lo observado en patología inflamatoria como en el eccema. Por otro lado, en cuanto a las reacciones irritativas, el patrón más indicativo sería el «patrón del poro», acompañado de eritema perifolicular. Estos hallazgos dermatoscópicos pueden ser de utilidad al clínico en su toma de decisiones ante una reacción dudosa (AU)

Toxicology assessment of manganese oxide nanomaterials with enhanced electrochemical properties using human in vitro models representing different exposure routes.

In the present study, a comparative human toxicity assessment between newly developed Mn3O4 nanoparticles with enhanced electrochemical properties (GNA35) and their precursor material (Mn3O4) was performed, employing different in vitro cellular models representing main exposure routes (inhalation, intestinal and dermal contact), namely the human alveolar carcinoma epithelial cell line (A549), the human colorectal adenocarcinoma cell line (HT29), and the reconstructed 3D human epidermal model EpiDerm. The obtained results showed that Mn3O4 and GNA35 harbour similar morphological characteristics, whereas differences were observed in relation to their surface area and electrochemical properties. In regard to their toxicological properties, both nanomaterials induced ROS in the A549 and HT29 cell lines, while cell viability reduction was only observed in the A549 cells. Concerning their skin irritation potential, the studied nanomaterials did not cause a reduction of the skin tissue viability in the test conditions nor interleukin 1 alpha (IL- 1 α) release. Therefore, they can be considered as not irritant nanomaterials according to EU and Globally Harmonized System of Classification and Labelling Chemicals. Our findings provide new insights about the potential harmful effects of Mn3O4 nanomaterials with different properties, demonstrating that the hazard assessment using different human in vitro models is a critical aspect to increase the knowledge on their potential impact upon different exposure routes.

ISO 10993-23 In vitro irritation testing for medical devices: Substantiating applicability to mild irritants and non-extractables.

Irritation testing is an integral part of the biocompatibility assessment of medical devices and has historically been conducted on animals, either by direct contact or with polar and non-polar solvent extracts. In 2018 an ISO-sponsored interlaboratory validation study demonstrated that two reconstituted human epidermis (RhE) based assays, which were adapted from validated methods used for industrial chemicals, produced results essentially equivalent to those obtained with in vivo tests. This led to the publication of the ISO 10993-23:2021 standard on irritation testing, which states that RhE-based assays are now the preferred method. The 2018 validation study evaluated strong irritants, so we tested nine mild irritants (GHS Category 3), neat and spiked at different concentrations into medical device extracts, per ISO 10993-23:2021. The results substantiated the applicability of RhE-based assays for evaluating mild irritants in medical device extracts. Moreover, the 2018 validation study tested solid extractable medical device materials but did not consider non-extractable medical device materials (e.g., creams, gels, or sprays). By testing nine marketed non-extractable materials, either neat or spiked with irritants, we also confirmed that RhE-based assays are readily applicable to such medical device materials.

Open-source human skin model with an in vivo-like barrier for drug testing.

There is a global trend towards the development of physiologically relevant in vitro skin models to reduce or replace animal testing in the evaluation of therapeutic drug candidates. However, only commercial reconstructed human epidermis models (RHEm) have undergone formal validation. Although these commercial models are suitable for a wide range of applications, they are costly, lack flexibility, and the protocols used to generate them are not transparent. In this study, we present an open-source full-thickness skin model (FTSm) and assess its potential for drug testing. The FTSm was developed using endogenous extracellular matrix to recreate the dermal compartment, avoiding animal-derived hydrogels. An RHEm based on an open-source protocol was evaluated in parallel. The integrity of the skin barrier was analyzed by challenging the surface with detergents and measuring cell viability as well as by trans-epithelial electrical resistance (TEER) measurements. Skin irritation studies were performed based on OECD guidelines and complemented with an evaluation of the impact on the skin barrier by TEER measurement. The permeation of a dye through the developed models and a commercial membrane (Strat-M®) was compared using Franz diffusion cells and an infinite dose approach. The FTSm demonstrated structural and barrier properties comparable to native human skin. Although the RHEm showed a better performance in drug testing, the FTSm presented better barrier properties than commercial models as reported in the literature. These skin models can be a valuable contribution to accelerating the development and dissemination of alternatives to animal testing, avoiding the limitations of commercial models.

Evaluation of Two Novel Hydantoin Derivatives Using Reconstructed Human Skin Model EpiskinTM: Perspectives for Application as Potential Sunscreen Agents.

This study aimed to assess two novel 5-arylideneimidazolidine-2,4-dione (hydantoin) derivatives (JH3 and JH10) demonstrating photoprotective activity using the reconstructed human skin model EpiskinTM. The skin permeability, irritation, and phototoxicity of the compounds was evaluated in vitro. Moreover, the in vitro genotoxicity and human metabolism of both compounds was studied. For skin permeation and irritation experiments, the test compounds were incorporated into a formulation. It was shown that JH3 and JH10 display no skin irritation and no phototoxicity. Both compounds did not markedly enhance the frequency of micronuclei in CHO-K1 cells in the micronucleus assay. Preliminary in vitro studies with liver microsomes demonstrated that hydrolysis appears to constitute their important metabolic pathway. EpiskinTM permeability experiments showed that JH3 permeability was lower than or close to currently used UV filters, whereas JH10 had the potential to permeate the skin. Therefore, a restriction of this compound permeability should be obtained by choosing the right vehicle or by optimizing it, which should be addressed in future studies.

The fabrication and assessment of mosquito repellent cream for outdoor protection.

Mosquito-borne infections like dengue, malaria, chikungunya, etc. are a nuisance and can cause profound discomfort to people. Due to the objectional side effects and toxicity associated with synthetic pyrethroids, N,N-diethyl-3-methylbenzamide (DEET), N,N-diethyl phenylacetamide (DEPA), and N,N-di ethyl benzamide (DEBA) based mosquito repellent products, we developed an essential oil (EO) based mosquito repellent cream (EO-MRC) using clove, citronella and lemongrass oil. Subsequently, a formulation characterization, bio-efficacy, and safety study of EO-MRC were carried out. Expression of Anti-OBP2A and TRPV1 proteins on mosquito head parts were studied by western blotting. In-silico screening was also conducted for the specific proteins. An FT-IR study confirmed the chemical compatibility of the EOs and excipients used in EO-MRC. The thermal behaviour of the best EOs and their mixture was characterized by thermogravimetric analysis (TGA). GC-MS examination revealed various chemical components present in EOs. Efficacy of EO-MRC was correlated with 12% N,N-diethyl benzamide (DEBA) based marketed cream (DBMC). Complete protection time (CPT) of EO-MRC was determined as 228 min. Cytotoxicity study on L-132 cell line confirmed the non-toxic nature of EO-MRC upon inhalation. Acute dermal irritation study, acute dermal dose toxicity study, and acute eye irritation study revealed the non-toxic nature of EO-MRC. Non-target toxicity study on Danio rerio confirmed EO-MRC as safer for aquatic non-target animals. A decrease in the concentration of acetylcholinesterase (AChE) was observed in transfluthrin (TNSF) exposed Wistar rats. While EO-MRC did not alter the AChE concentrations in the exposed animals. Results from western blotting confirmed that Anti-OBP2A and TRPV1 proteins were inhibited in TNSF exposed mosquitoes. Mosquitoes exposed to EO-MRC showed a similar expression pattern for Anti-OBP2A and TRPV1 as the control group. In silico study revealed eight identified compounds of the EOs play significant roles in the overall repellency property of the developed product. The study emphasizes the mosquito repellent activity of EO-MRC, which could be an effective, eco-friendly, and safer alternative to the existing synthetic repellents for personal protection against mosquitoes during field conditions.

In Vitro and Clinical Evaluation of Cannabigerol (CBG) Produced via Yeast Biosynthesis: A Cannabinoid with a Broad Range of Anti-Inflammatory and Skin Health-Boosting Properties.

Cannabigerol (CBG) is a minor non-psychoactive cannabinoid present in Cannabis sativa L. (C. sativa) at low levels (<1% per dry weight) that serves as the direct precursor to both cannabidiol (CBD) and tetrahydrocannabinol (THC). Consequently, efforts to extract and purify CBG from C. sativa is both challenging and expensive. However, utilizing a novel yeast fermentation technology platform, minor cannabinoids such as CBG can be produced in a more sustainable, cost-effective, and timely process as compared to plant-based production. While CBD has been studied extensively, demonstrating several beneficial skin properties, there are a paucity of studies characterizing the activity of CBG in human skin. Therefore, our aim was to characterize and compare the in vitro activity profile of non-psychoactive CBG and CBD in skin and be the first group to test CBG clinically on human skin. Gene microarray analysis conducted using 3D human skin equivalents demonstrates that CBG regulates more genes than CBD, including several key skin targets. Human dermal fibroblasts (HDFs) and normal human epidermal keratinocytes (NHEKs) were exposed in culture to pro-inflammatory inducers to trigger cytokine production and oxidative stress. Results demonstrate that CBG and CBD reduce reactive oxygen species levels in HDFs better than vitamin C. Moreover, CBG inhibits pro-inflammatory cytokine (Interleukin-1ß, -6, -8, tumor necrosis factor α) release from several inflammatory inducers, such as ultraviolet A (UVA), ultraviolet B (UVB), chemical, C. acnes, and in several instances does so more potently than CBD. A 20-subject vehicle-controlled clinical study was performed with 0.1% CBG serum and placebo applied topically for 2 weeks after sodium lauryl sulfate (SLS)-induced irritation. CBG serum showed statistically significant improvement above placebo for transepidermal water loss (TEWL) and reduction in the appearance of redness. Altogether, CBG's broad range of in vitro and clinical skin health-promoting activities demonstrates its strong potential as a safe, effective ingredient for topical use and suggests there are areas where it may be more effective than CBD.

Synthesis and biological evaluation of ruthenium polypyridine complexes with 18ß-glycyrrhetinic acid as antibacterial agents against Staphylococcus aureus.

Four new ruthenium(II) polypyridine complexes bearing 18ß-glycyrrhetinic acid derivatives, [Ru(bpy)2L](PF6)2 (Ru1), [Ru(dmb)2L](PF6)2 (Ru2), [Ru(dtb)2L](PF6)2 (Ru3) and [Ru(phen)2L](PF6)2 (Ru4) (bpy = 2,2-bipyridine, dmb = 4,4'-dimethyl-2,2'-bipyridine, dtb = 4,4'-di-tert-butyl-2,2'-bipyridine, phen = 1,10-phenanthroline and L is the GA modified new ligand) were designed and synthesized. Their antimicrobial activities against Staphylococcus aureus (S. aureus) were evaluated and all complexes showed an obvious inhibitory effect, especially, the minimum inhibitory concentration (MIC) value of Ru2 was 3.9 µg mL-1. Moreover, Ru2 was found to significantly inhibit the formation of biofilms. The membrane-compromising action mode was suggested to be their potential antibactericidal mechanism. In hemolysis experiments, Ru2 hardly showed cytotoxicity to mammalian erythrocytes. Furthermore, the synergism between Ru2 and common antibiotics, such as ampicillin, chloramphenicol, tetracyclines and ofloxacin, against S. aureus was also detected using the checkerboard method. Finally, a mouse skin infection model was established to evaluate the antibacterial activity of Ru2in vivo, and the results showed that Ru2 could effectively promote wound healing in mice infected with S. aureus. Moreover, the results of histopathological research were consistent with the results of the hemolysis test, indicating that the Ru2 complex was almost non-toxic. Thus, it was demonstrated that the polypyridine ruthenium complexes modified with glycyrrhetinic acid (GA) are a promising strategy for developing interesting antibacterial agents.

A hypothetical skin sensitisation next generation risk assessment for coumarin in cosmetic products.

Next generation Risk Assessment (NGRA) is an exposure-led, hypothesis-driven approach which integrates new approach methodologies (NAMs) to assure safety without generating animal data. This hypothetical skin allergy risk assessment of two consumer products - face cream containing 0.1% coumarin and deodorant containing 1% coumarin - demonstrates the application of our skin allergy NGRA framework which incorporates our Skin Allergy Risk Assessment (SARA) Model. SARA uses Bayesian statistics to provide a human relevant point of departure and risk metric for a given chemical exposure based upon input data that can include both NAMs and historical in vivo studies. Regardless of whether NAM or in vivo inputs were used, the model predicted that the face cream and deodorant exposures were low and high risk respectively. Using only NAM data resulted in a minor underestimation of risk relative to in vivo. Coumarin is a predicted pro-hapten and consequently, when applying this mechanistic understanding to the selection of NAMs the discordance in relative risk could be minimized. This case study demonstrates how integrating a computational model and generating bespoke NAM data in a weight of evidence framework can build confidence in safety decision making.

Glimepiride-Loaded Nanoemulgel; Development, In Vitro Characterization, Ex Vivo Permeation and In Vivo Antidiabetic Evaluation.

Glimepiride (GMP), an oral hypoglycemic agent is extensively employed in the treatment of type 2 diabetes. Transdermal delivery of GMP has been widely investigated as a promising alternative to an oral approach but the delivery of GMP is hindered owing to its low solubility and permeation. The present study was designed to formulate topical nanoemulgel GMP system and previously reported solubility enhanced glimepiride (GMP/ßCD/GEL-44/16) in combination with anti-diabetic oil to enhance the hypoglycemic effect. Nanoemulsions were developed using clove oil, Tween-80, and PEG-400 and were gelled using xanthan gum (3%, w/w) to achieve the final nanoemulgel formulations. All of the formulations were evaluated in terms of particle size, zeta potential, pH, conductivity, viscosity, and in vitro skin permeation studies. In vivo hypoglycemic activity of the optimized nanoemulgel formulations was evaluated using a streptozocin-induced diabetes model. It was found that a synergistic combination of GMP with clove oil improved the overall drug permeation across the skin membrane and the hypoglycemic activity of GMP. The results showed that GMP/ßCD/GEL-44/16-loaded nanoemulgel enhanced the in vitro skin permeation and improved the hypoglycemic activity in comparison with pure and marketed GMP. It is suggested that topical nano emulsion-based GMP gel and GMP/ßCD/GEL-44/16 could be an effective alternative for oral therapy in the treatment of diabetes.

Topical Delivery of Geranium/Calendula Essential Oil-Entrapped Ethanolic Lipid Vesicular Cream to Combat Skin Aging.

The present study deals with the evaluation of the age-defying potential of topical cream formulations bearing Geranium essential oil/Calendula essential oil-entrapped ethanolic lipid vesicles (ELVs). Two types of cream formulations were prepared, viz., conventional and ELVs spiked o/w creams. Essential oil- (EO-) loaded ELVs were characterized by vesicle size, polydispersity index, encapsulation efficiency, and scanning electron microscopy. The cream formulations were evaluated for homogeneity, spreadability, viscosity, pH, in vitro antioxidant capacity, sun protection factor, and in vitro collagenase and elastase inhibition capacity. Confocal laser scanning microscopy (CLSM) was performed to ascertain skin permeation of conventional and vesicular cream. The results of in vitro antioxidant studies showed that GEO-/CEO-loaded vesicular creams have notable antioxidant capacity when compared to nonvesicular creams. GEO- or CEO-loaded vesicular creams exhibited the highest SPF value 10.26 and 18.54, respectively. Both the EO-based vesicular creams showed in vitro collagenase and elastase enzyme inhibition capacity. CLSM images clearly depicted that vesicular cream deep into the skin layers. From the research findings, the age-defying potential and photoprotective effects of GEO and CEO were confirmed. It can be concluded that ELVs are able to preserve the efficiency of EOs and have the potential to combat skin aging.

Tandem mass spectrometry of aqueous extract from Ficus dubia sap and its cell-based assessments for use as a skin antioxidant.

Since 2006, Ficus dubia has been reported as a new Ficus species in Thailand. As per our recent report, the red-brown aqueous extract of F. dubia sap (FDS) has been determined to strongly exhibit in vitro anti-radicals. However, the phytochemicals in the FDS extract related to health-promoting antioxidation have not been explored. Thus, in this study, we aimed to investigate the chemical components of the F. dubia sap extract by liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI-MS/QTOF-MS) and its potential use in cosmetics in terms of cellular antioxidation on keratinocytes (HaCaT), phototoxicity, and irritation on 3D skin cell models following standard tests suggested by the Organization for Economic Cooperation and Development (OECD). It was found that the sap extract was composed of quinic acid and caffeoyl derivatives (e.g., syringoylquinic acid, 3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, and dimeric forms of caffeoylquinic acids). The extract has significantly exhibited antioxidant activity against H2O2-induced oxidative stress in HaCaT cells. The cellular antioxidative effect of the FDS extract was remarkably dependent on the presence of 3- and 4-O-caffeoylquinic acid in the extract. Furthermore, the FDS extract showed negative results on skin phototoxicity and irritation. Overall, the results reveal that the FDS extract could be developed as a new antioxidant candidate for a skin healthcare product.

Ex Vivo Human Skin: An Alternative Test System for Skin Irritation and Corrosion Assays.

Native human skin has been reported in the literature as being an important experimental model for studying skin biology. Studies performed by our group have shown that ex vivo skin, from elective plastic surgery, maintains the biological characteristics of native skin under specific culture conditions. As such, it might be a feasible model for the safety and efficacy testing of topical substances. While Brazil is at the forefront of global regulation implementation, Brazilian researchers are not always able to transfer certain widely used protocols to their laboratories, particularly protocols that involve the use of reconstructed tissues with limited viability, such as those for skin corrosion (OECD TG 431) and irritation testing (OECD TG 439). In this study, we investigated the applicability of the ex vivo skin model to the evaluation of irritation and corrosion potential of a number of proficiency substances described in TG 431 and TG 439. The skin fragments were standardised in size and diameter, and placed into cell culture inserts. The experimental protocol was conducted according to TG 431 and TG 439. The results obtained show that ex vivo skin could represent a promising tool for the evaluation of irritation and corrosion potential of substances (subject to inclusion and exclusion criteria), as recommended by OECD guidelines. While this is a proof-of-concept study, the use of ex vivo skin should be considered for such testing.

Nanoparticle impregnated self-supporting protein gel for enhanced reduction in oxidative stress: A molecular dynamics insight for lactoferrin-polyphenol interaction.

In the present work, lactoferrin (Lf) based nanoparticle incorporated self-supporting gel encapsulating a flavonoid, quercetin (Q), was developed. The complex formation between Lf and Q was assessed using molecular docking and dynamics simulation that lactoferrin and quercetin showed strong interaction and binding supporting hydrophobic interaction. The microscopic, spectroscopic, and x-ray techniques were used to characterize the gel extensively. In vitro drug release was studied to understand the release pattern of quercetin from the protein gel. The viscosity of the gel and its rheological characteristics were determined using a Brookfield viscometer. Ex vivo skin permeation studies using vertical diffusion cells were carried out to understand its skin permeation properties. The gel showed strong anti-oxidant activity using the DPPH scavenging assay. The enhanced effect of the Lf-Q complex on antioxidant enzyme activity (superoxide dismutase, catalase, and malondialdehyde), was supported by molecular dynamics, surface hydrophobicity, and in vitro studies. To investigate the effect of the gel on angiogenesis, the chorioallantoic membrane assay was performed and its compatibility with erythrocytes was also assessed. Suitability for topical administration was assessed using skin irritation studies performed on Sprague Dawley rats. The overall results suggest that the developed NiPG is suitable for cutaneous localization of quercetin with enhanced antioxidant activity.