Circadian Oscillations in Skin and Their Interconnection with the Cycle of Life.

Periodically oscillating biological processes, such as circadian rhythms, are carefully concerted events that are only beginning to be understood in the context of tissue pathology and organismal health, as well as the molecular mechanisms underlying these interactions. Recent reports indicate that light can independently entrain peripheral circadian clocks, challenging the currently prevalent hierarchical model. Despite the recent progress that has been made, a comprehensive overview of these periodic processes in skin is lacking in the literature. In this review, molecular circadian clock machinery and the factors that govern it have been highlighted. Circadian rhythm is closely linked to immunological processes and skin homeostasis, and its desynchrony can be linked to the perturbation of the skin. The interplay between circadian rhythm and annual, seasonal oscillations, as well as the impact of these periodic events on the skin, is described. Finally, the changes that occur in the skin over a lifespan are presented. This work encourages further research into the oscillating biological processes occurring in the skin and lays the foundation for future strategies to combat the adverse effects of desynchrony, which would likely have implications in other tissues influenced by periodic oscillatory processes.

Lateral Dermal Penetration Is Dependent on the Lipophilicity of Active Ingredients.

INTRODUCTION: With its large surface area, skin facilitates a topical administration of active ingredients, and thus percutaneous delivery to a specific target site. Due to its high barrier function and different diffusion characteristics, skin governs the efficacy of these active ingredients and a bioavailability in the epidermal and dermal tissue. OBJECTIVE: In order to characterize the vertical and lateral movement of molecules into and inside the skin, the diffusivity of active ingredients with different physicochemical properties and their penetration ability in different dermal skin layers was investigated. METHODS: A novel lateral dermal microdialysis (MD) penetration setup was used to compare the diffusion characteristics of active ingredients into superficial and deep-implanted MD membranes in porcine skin. The corresponding membrane depth was determined via ultrasound and the active ingredients concentration via high-pressure liquid chromatography measurement. RESULTS: The depth depended penetration of superficial and deep-implanted MD membranes and the quantitative diffusivity of two active ingredients was compared. An experimental lateral MD setup was used to determine the influence of percutaneous skin penetration characteristics of an active ingredient with different lipophilic and hydrophilic characteristics. Therefore, hydrophilic caffeine and lipophilic LIP1, which have an identical molecular weight but different lipophilic characteristics, were tested for their penetration ability inside a propylene glycol and oleic acid formulation. CONCLUSION: The vertical and lateral penetration movement of caffeine was found to exceed that of LIP1 through the hydrophilic dermal environment. The findings of this study show that the lipophilicity of active ingredients influences the penetration movement and that skin enables a conical increasing lateral diffusivity and transdermal delivery.

Methoxy-Monobenzoylmethane Protects Skin from UV-Induced Damages in a Randomized, Placebo Controlled, Double-Blinded Human In Vivo Study and Prevents Signs of Inflammation While Improving the Skin Barrier.

INTRODUCTION: Sun protection is important in skin care and requires special attention as inefficient protection might trigger skin pathologies including polymorphic light eruption (PLE). The reduce-improve-protect (RIP) concept to avoid the onset of ultraviolet (UV) irradiation-induced diseases or damage to human skin is important. Methoxy-monobenzoylmethane (MeO-MBM), which is neither a UVB nor a UVA filter, converts to the UV filter avobenzone under UV irradiation and further acts as a photoantioxidant during its conversion process and initially as an antioxidant material. The aim of this study was to understand the mechanisms by which MeO-MBM improves the condition of UV-stressed skin through its photoantioxidant properties. The improvement of the skin condition by the activity of MeO-MBM as active ingredient was also investigated. METHODS: Potential molecular targets were identified by in silico docking to numerous cellular membrane receptors on the cell surface or nuclear membrane, followed by microarray analysis of 164 genes after MeO-MBM treatment of normal human epidermal keratinocytes (NHEK). We conducted randomized, double-blinded, intra-individual comparison vs. placebo studies on ten volunteers, aged between 34 and 65 years, to assess the effect of MeO-MBM in vivo. The effect after UV-induced inflammation was assessed in a protective and curative set-up with 2% MeO-MBM vs. 1% hydrocortisone and placebo based on the change in blood flow. The barrier function of the skin was assessed by the change in transepidermal water loss (TEWL), skin scaling and skin thickness after the treatment with MeO-MBM. Additionally, the effect of MeO-MBM after UV-induced stress on the activation of ferritin in human explants was determined ex vivo. RESULTS: A docking simulation of MeO-MBM showed a potential interaction with the retinoic acid receptor gamma and further revealed downregulation of proteins related to inflammation. In the protective treatment set-up, after 24 h MeO-MBM significantly reduced the delta blood flow compared to placebo, while this reduction was more prominent with hydrocortisone. In the curative treatment set-up, a greater reduction in delta blood flow was also observed with MeO-MBM compared to placebo and similar to hydrocortisone. Treatment with MeO-MBM revealed an improvement in skin barrier function as a result of decreased TEWL, reduced skin scaling and increased skin thickness. Immunohistochemistry staining of ferritin on human skin explants further showed that the treatment with MeO-MBM reduced the ferritin expression. CONCLUSION: Based on these results, MeO-MBM is capable of exerting an anti-aging activity via the retinoic acid receptor gamma. Its anti-inflammatory and anti-oxidative activity manifested via the downregulation of multiple anti-inflammatory genes as well as the reduction of ferritin in skin tissue. This study shows that the multidimensional functionality of MeO-MBM offers an effective approach to combat acute and chronic deleterious effects of oxidative UV damage while simultaneously enhancing the skin barrier function.

Activity-Guided Characterization of COX-2 Inhibitory Compounds in Waltheria indica L. Extracts.

Inflammation is the body's response to infection or tissue injury in order to restore and maintain homeostasis. Prostaglandin E2 (PGE-2) derived from arachidonic acid (AA), via up-regulation of cyclooxygenase-2 (COX-2), is a key mediator of inflammation and can also be induced by several other factors including stress, chromosomal aberration, or environmental factors. Targeting prostaglandin production by inhibiting COX-2 is hence relevant for the successful resolution of inflammation. Waltheria indica L. is a traditional medicinal plant whose extracts have demonstrated COX-2 inhibitory properties. However, the compounds responsible for the activity remained unknown. For the preparation of extracts with effective anti-inflammatory properties, characterization of these substances is vital. In this work, we aimed to address this issue by characterizing the substances responsible for the COX-2 inhibitory activity in the extracts and generating prediction models to quantify the COX-2 inhibitory activity without biological testing. For this purpose, an extract was separated into fractions by means of centrifugal partition chromatography (CPC). The inhibitory potential of the fractions and extracts against the COX-2 enzyme was determined using a fluorometric COX-2 inhibition assay. The characterizations of compounds in the fractions with the highest COX-2 inhibitory activity were conducted by high resolution mass spectrometry (HPLC-MS/MS). It was found that these fractions contain alpha-linolenic acid, linoleic acid and oleic acid, identified and reported for the first time in Waltheria indica leaf extracts. After analyzing their contents in different Waltheria indica extracts, it could be demonstrated that these fatty acids are responsible for up to 41% of the COX-2 inhibition observed with Waltheria indica extract. Additional quantification of secondary metabolites in the extract fractions revealed that substances from the group of steroidal saponins and triterpenoid saponins also contribute to the COX-2 inhibitory activity. Based on the content of compounds contributing to COX-2 inhibition, two mathematical models were successfully developed, both of which had a root mean square error (RMSE) = 1.6% COX-2 inhibitory activity, demonstrating a high correspondence between predicted versus observed values. The results of the predictive models further suggested that the compounds contribute to COX-2 inhibition in the order linoleic acid > alpha linolenic acid > steroidal saponins > triterpenoid saponins. The characterization of substances contributing to COX-2 inhibition in this study enables a more targeted development of extraction processes to obtain Waltheria indica extracts with superior anti-inflammatory properties.

Methoxy-Monobenzoylmethane Protects Human Skin against UV-Induced Damage by Conversion to Avobenzone and Radical Scavenging.

Avobenzone, one of the most commonly used UV filters in topical sunscreens, is susceptible to photodegradation with a consequential reduction of its UV absorbing properties. This loss of function may lead to skin irritation, photodermatosis, and photoallergic reactions caused by photodegradation byproducts. In this work, we aim to address this issue with a substance named methoxy-monobenzoylmethane (MeO-MBM), which is neither a UVB nor a UVA filter, but which converts to avobenzone, a known and approved UVA filter, under mainly UVB light irradiation. The antioxidant and intracellular radical formation properties of MeO-MBM were compared to the ones of avobenzone. The UV irradiation of MeO-MBM led to an increase in UV absorption primarily in the UVA range after conversion, both in vitro and in vivo. HPTLC and UHPLC studies illustrate the conversion of MeO-MBM to avobenzone in vitro after irradiation at 250 kJ/m2, reaching a conversion rate of 48.8%. A stable molecular antioxidant activity was observed, since 100-µM MeO-MBM was measured to be 11.2% in the DPPH assay, with a decrease to 9.7% after irradiation. In comparison, the molecular antioxidant activity of 100-µM avobenzone was determined to be 0.8%. In keratinocytes, MeO-MBM reduces the intracellular ROS by 90% and avobenzone by 75% with tBHP as the inducer and by 53% and 57%, respectively, when induced by pyocyanin, indicating the redox scavenging capacity of both these molecules. These results indicate that MeO-MBM functions initially as an antioxidant material and as a photoantioxidant during its conversion process to avobenzone. This research provides insight into the development of active ingredients for topical applications with dynamic functionalities. Using this approach, we demonstrate the possibility to extend the UV protection offered to skin cells while combating cellular stress in parallel.

A Cornflower Extract Containing N-Feruloylserotonin Reduces Inflammation in Human Skin by Neutralizing CCL17 and CCL22 and Inhibiting COX-2 and 5-LOX.

Thymus and Activation-Regulated Chemokine (TARC/CCL17) and Macrophage-Derived Chemokine (MDC/CCL22) are two key chemokines exerting their biological effect via binding and activating a common receptor CCR4, expressed at the surface of type 2 helper T (Th2) cells. By recruiting Th2 cells in the dermis, CCL17 and CCL22 promote the development of inflammation in atopic skin. The aim of this research was to develop a plant extract whose biological properties, when applied topically, could be beneficial for people with atopic-prone skin. The strategy which was followed consisted in identifying ligands able to neutralize the biological activity of CCL17 and CCL22. Thus, an in silico molecular modeling and a generic screening assay were developed to screen natural molecules binding and blocking these two chemokines. N-Feruloylserotonin was identified as a neutraligand of CCL22 in these experiments. A cornflower extract containing N-feruloylserotonin was selected for further in vitro tests: the gene expression modulation of inflammation biomarkers induced by CCL17 or CCL22 in the presence or absence of this extract was assessed in the HaCaT keratinocyte cell line. Additionally, the same cornflower extract in another vehicle was evaluated in parallel with N-feruloylserotonin for cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) enzymatic cellular inhibition. The cornflower extract was shown to neutralize the two chemokines in vitro, inhibited COX-2 and 5-LOX, and demonstrated anti-inflammatory activities due mainly to the presence of N-feruloylserotonin. Although these findings would need to be confirmed in an in vivo study, the in vitro studies lay the foundation to explain the benefits of the cornflower extract when applied topically to individuals with atopic-prone skin.

Comparison of Membrane Depth Determination Techniques for Active Ingredient Skin Penetration Studies Using Microdialysis.

INTRODUCTION: The skin is a major physical barrier to the environment, and thus, percutaneous delivery of active ingredients to the dermal target site faces a unique set of hurdles. The efficacy of these active ingredients is governed by their release into the underlying epidermal and dermal tissue, especially when administered topically. OBJECTIVE: The aim of this study was to understand if different physicochemical properties influence the skin penetration of active ingredients and the depth to which they penetrate into the dermis. METHODS: A microdialysis (MD) setup was used to compare the percutaneous penetration in superficial and deep implanted MD membranes in porcine skin. The precise MD membrane depth was determined using histological sectioning paired with microscopy, ultrasound, and a novel computed tomographic approach. RESULTS: In study A, the measured depth of the superficial and deep implanted MD membranes was compared using histological sectioning, ultrasound, and computed tomography. Experimental determination of the depth up to which penetration occurs was found to be crucial to percutaneous penetration studies. In study B, the lipophilic differences of the active ingredients and its influences on the penetration was tested using hydrophilic caffeine and lipophilic LIP1 as model compounds, which have an identical molecular weight with different lipophilic characteristics. It is assumed that the lipophilic characteristics of active ingredients influence their penetration and thus governs the concentration of these molecules reaching their target site. CONCLUSION: The transdermal penetration of caffeine was found to exceed that of LIP1 through the hydrophilic environment of the dermis. Thus, the findings of this study show that the precise MD dermis localization and the physicochemical properties, such as lipophilicity, influence the penetration rate of active ingredients and lay the foundation for creating optimized transdermal delivery systems.

Retaining Skin Barrier Function Properties of the Stratum Corneum with Components of the Natural Moisturizing Factor-A Randomized, Placebo-Controlled Double-Blind In Vivo Study.

The influence of a topically applied formulation containing components of natural moisturizing factor (NMF) on barrier-related parameters of the stratum corneum (SC) was investigated in vivo using confocal Raman microspectroscopy in a randomized, placebo-controlled double-blind study on 12 volunteers for 14 days. This method allowed for the elucidation of subtle differences between the verum and the placebo even though the components of the verum naturally occur in the SC. This differentiation is not possible non-invasively by conventional methods. In this study, we found that the applied verum and placebo formulations disrupted the equilibrium of water, NMF and lipids in the SC. The adverse effects of the formulation could be mitigated by incorporating it into a simplified supplementation of NMF molecules. As a long-term effect, the amount of strongly bound water increases at 30-40% SC depth (p < 0.05) and the amount of weakly bound water decreases at 30-40% SC depth (p < 0.05) for the verum. This supplement was also unexpectedly able to prevent intercellular lipids (ICL) disorganization in selected depths. In the long term, the verum treatment limited the lateral disorganization of the ICL to the upper 20% SC depth. Further research is required to elucidate the interplay of these factors in the SC, to better understand their contribution to the equilibrium and barrier function of the skin. This understanding of the interaction of these naturally occurring components could help in the future to develop and optimize topical treatments for diseases like psoriasis, atopic dermatitis, ichthyosis where the skin barrier is disrupted.

Identification of plant metabolite classes from Waltheria Indica L. extracts regulating inflammatory immune responses via COX-2 inhibition.

ETHNOPHARMACOLOGICAL RELEVANCE: Waltheria Indica L. is traditionally used in Africa, South America and Hawaii to treat pain, anemia, diarrhea, epilepsy and inflammatory related diseases. AIM OF THE STUDY: This study aimed to identify extraction parameters to maximize tiliroside yield and to quantitative secondary metabolite composition of Waltheria Indica under various extraction conditions. The extracts were tested for COX-2 inhibition and their activity correlated with the type and quantity of the secondary metabolites. Insight was gained about how extraction parameters influence the extract composition and thus the COX-2 enzymatic inhibitory activity. MATERIALS AND METHODS: Powdered leaves of Waltheria Indica were extracted using water, methanol, ethyl acetate and ethanol at different temperatures. Tiliroside was identified by HPLC-HRMS n and quantified using a tiliroside standard. The compound groups of the secondary metabolites were quantified by spectrometric methods. Inhibitory potential of different Waltheria extracts against the COX-2 enzyme was determined using a fluorometric COX-2 inhibition assay. RESULTS: The molecule, tiliroside, exhibited a COX-2 inhibition of 10.4% starting at a concentration of 15 µM and increased in a dose dependent manner up to 51.2% at 150 µM. The ethanolic extract at 30 °C and the ethyl acetate extract at 90 °C inhibited COX-2 with 37.7% and 38.9%, while the methanolic and aqueous extract showed a lower inhibition of 21.9% and 9.2% respectively. The results concerning phenol, alkaloid and tiliroside concentration in the extracts showed no dependence on COX-2 inhibition. The extracts demonstrated a direct correlation of COX-2 inhibitory activity with their triterpenoid-/steroidal-saponin concentration. COX-2 inhibition increased linearly with the concentration of the saponins. CONCLUSION: The data suggest that Waltheria Indica extracts inhibit the key inflammatory enzyme, COX-2, as a function of triterpenoid- and steroidal-saponin concentration and support the known efficacy of extracted Waltheria Indica leaves as a traditional treatment against inflammation related diseases.

Amino acids in the cultivation of mammalian cells.

Amino acids are crucial for the cultivation of mammalian cells. This importance of amino acids was realized soon after the development of the first cell lines, and a solution of a mixture of amino acids has been supplied to cultured cells ever since. The importance of amino acids is further pronounced in chemically defined mammalian cell culture media, making the consideration of their biological and chemical properties necessary. Amino acids concentrations have been traditionally adjusted to their cellular consumption rates. However, since changes in the metabolic equilibrium of amino acids can be caused by changes in extracellular concentrations, metabolomics in conjunction with flux balance analysis is being used in the development of culture media. The study of amino acid transporters is also gaining importance since they control the intracellular concentrations of these molecules and are influenced by conditions in cell culture media. A better understanding of the solubility, stability, dissolution kinetics, and interactions of these molecules is needed for an exploitation of these properties in the development of dry powdered chemically defined media for mammalian cells. Due to the complexity of these mixtures however, this has proven to be challenging. Studying amino acids in mammalian cell culture media will help provide a better understanding of how mammalian cells in culture interact with their environment. It would also provide insight into the chemical behavior of these molecules in solutions of complex mixtures, which is important in the understanding of the contribution of individual amino acids to protein structure.