Skin Penetration of Epigallocatechin-3-Gallate and Quercetin from Green Tea and Ginkgo biloba Extracts Vehiculated in Cosmetic Formulations

Green tea (Camellia sinensis) and Ginkgo biloba extracts in cosmetic formulations have been suggested to protect the skin against UV-induced damage and skin ageing. Thus, it is very important to assess the human skin penetration of their major flavonoids to verify if they penetrate and remain in the skin to exert their proposed effects. The aim of this study was to evaluate the human skin penetration of epigallocatechin-3-gallate (EGCG) and quercetin from green tea and G. biloba extracts vehiculated in cosmetic formulations. This study was conducted with fresh dermatomed human Caucasian skin from abdominal surgery mounted on static Franz diffusion cells. Skin samples were mounted between two diffusion half-cells and 10 mg/cm(2) of formulations supplemented with 6% of green tea or G. biloba extract were applied on the skin surface. The receptor fluid was removed after 6 and 24 h and analyzed by high-performance liquid chromatography for the quantification of the flavonoids. The stratum corneum was removed by tape stripping and immersed in methanol and the epidermis was mechanically separated from the dermis and triturated in methanol to extract EGCG and quercetin. The results showed that the flavonoids under study penetrated into the skin, without reaching the receptor fluid. The majority of EGCG was quantified in the stratum corneum (0.87 mu g/cm(2)), which was statistically higher than the EGCG concentrations found in viable epidermis (0.54 mu g/cm(2)) and in the dermis (0.38 mu g/cm(2)). The majority of quercetin was quantified in the viable epidermis (0.23 mu g/cm(2)), which was statistically higher than the EGCG concentration found in the stratum corneum layer (0.17 mu g/cm(2)). Finally, it can be concluded that EGCG and quercetin from green tea and G. biloba extracts vehiculated in cosmetic formulations presented good skin penetration and retention, which can favor their skin effects. Copyright (C) 2009 S. Karger AG, Basel

In vitro antioxidant activity and in vivo efficacy of topical formulations containing vitamin C and its derivatives studied by non-invasive methods

Background/purpose: Vitamins C and its derivatives, mainly due to their antioxidant properties, are being used in cosmetic products to protect and to reduce the signs of ageing. However, there are no studies comparing the effects of vitamin C [ascorbic acid (AA)] and its derivatives, magnesium ascorbyl phosphate (MAP) and ascorbyl tetra-isopalmitate (ATIP), when vehiculated in topical formulations, mainly using objective measurements, which are an important tool in clinical efficacy studies. Thus, the objective of this study was to determine the in vitro antioxidant activity of AA and its derivatives, MAP and ATIP, as well as their in vivo efficacy on human skin, when vehiculated in topical formulations. Methods: The study of antioxidant activity in vitro was performed with an aqueous and a lipid system. The in vivo methodology consisted of the application of these formulations on human volunteers` forearm skin and the analysis of the skin conditions after 4-week period daily applications in terms of transepidermal water loss (TEWL), stratum corneum moisture content and viscoelasticity using a Tewameter (R), Corneometer (R) and Cutometer (R), respectively. Results: In vitro experiments demonstrated that in an aqueous system, AA had the best antioxidant potential, and MAP was more effective than ATIP, whereas in the lipid system ATIP was more effective than MAP. In in vivo studies, all formulations enhanced stratum corneum moisture content after a 4-week period daily applications when compared with baseline values; however, only the formulation containing AA caused alterations in TEWL values. The formulations containing MAP caused alterations in the viscoelastic-to-elastic ratio, which suggested its action in the deeper layers of the skin. Conclusion: AA and its derivates presented an in vitro antioxidant activity but AA had the best antioxidant effect. In in vivo efficacy studies, only the formulation containing AA caused alterations in TEWL values and the formulation containing MAP caused alterations in the viscoelastic-to-elastic ratio. This way, vitamin C derivatives did not present the same effects of AA on human skin; however, MAP showed other significant effect-improving skin hydration, which is very important for the normal cutaneous metabolism and also to prevent skin alterations and early ageing.

A simple analysis of the changes during evaporation of a commercial emulsion of unknown composition

Optical microscopy and centrifugation were used to observe the Structural changes during evaporation of a commercial skin lotion of unknown composition. The degree of evaporation was determined from the changed weight of a microscope slide with the emulsion on a defined area and thickness, the evaporation loss versus time being measured by a balance under an infrared lamp. The results revealed not only which parts of the emulsion were most prone to evaporation without chemical analysis, but also gave surprising information as to which kind of structures would appear after extensive evaporation. The importance of these changes for the action of a skin lotion is briefly discussed.

Quercetin inhibits UV irradiation-induced inflammatory cytokine production in primary human keratinocytes by suppressing NF-kappa B pathway

Background: Topical flavonoids, such as quercetin, have been shown to reduce ultraviolet (UV) irradiation-mediated skin damage. However, the mechanisms and signaling pathways involved in this protective effect are not clear. UV irradiation leads to activation of two major signaling pathways, namely nuclear factor kappa B (NF-kappa B) and activator protein-1 (AP-1) pathways. Activation of NF-kappa B pathway by UV irradiation stimulates inflammatory cytokine expression, whereas activation of AP-1 pathway by UV irradiation promotes matrix metalloproteinase (MMP) production. Both pathways contribute to UV irradiation-induced skin damage, such as photoaging and skin tumor formation. Objective: To elucidate the underlying mechanism, we examined the effect of quercetin on UV irradiation induced activation of NF-kappa B and AP-1 pathways. Methods: Primary human keratinocytes, the major skin cell type subjected to physiological solar UV irradiation, were used to study the effects of quercetin on UV irradiation-induced signal transduction pathways. Results: Quercetin decreased UV irradiation-induced NF-kappa B DNA-binding by 80%. Consequently, quercetin suppressed UV irradiation-induced expression of inflammatory cytokines IL-1 beta (similar to 60%), IL-6 (similar to 80%), IL-8 (similar to 76%) and TNF-alpha (similar to 69%). In contrast, quercetin had no effect on UV irradiation activation of three MAP kinases, ERK, JNK, or p38. Accordingly, induction of AP-1 target genes such as MMP-1 and MMP-3 by UV irradiation was not suppressed by quercetin. Conclusion: Our data indicate that the ability of quercetin to block UV irradiation-induced skin inflammation is mediated, at least in part, by its inhibitory effect on NF-kappa B activation and inflammatory cytokine production. (C) 2011 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Biotype stability of Candida albicans isolates after culture storage determined by randomly amplified polymorphic DNA and phenotypical methods

P>Typing methods to evaluate isolates in relation to their phenotypical and molecular characteristics are essential in epidemiological studies. In this study, Candida albicans biotypes were determined before and after storage in order to verify their stability. Twenty C. albicans isolates were typed by Randomly Amplified Polymorphic DNA (RAPD), production of phospholipase and proteinase exoenzymes (enzymotyping) and morphotyping before and after 180 days of storage in Sabouraud dextrose agar (SDA) and sterilised distilled water. Before the storage, 19 RAPD patterns, two enzymotypes and eight morphotypes were identified. The fragment patterns obtained by RAPD, on the one hand, were not significantly altered after storage. On the other hand, the majority of the isolates changed their enzymotype and morphotype after storage. RAPD typing provided the better discriminatory index (DI) among isolates (DI = 0.995) and maintained the profile identified, thereby confirming its utility in epidemiological surveys. Based on the low reproducibility observed after storage in SDA and distilled water by morphotyping (DI = 0.853) and enzymotyping (DI = 0.521), the use of these techniques is not recommended on stored isolates.

A novel mutation in the L12 domain of keratin 5 in the Kobner variant of epidermolysis bullosa simplex

We have identified a novel mutation within the linker L12 region of keratin 5 (K5) in a family with the Kobner variant of epidermolysis bullosa simplex. The pattern of inheritance of the disorder in this family is consistent with an autosomal dominant mode of transmission. Affected individuals develop extensive and generalized blistering at birth or early infancy but in later years clinical manifestations are largely confined to palmo-plantar surfaces. Direct sequencing of polymerase chain reaction products revealed a T to C transition within codon 323 of K5 in affected individuals, resulting in a valine to alanine substitution of the seventh residue within the L12 linker domain. This mutation was not observed in unaffected family members or in 100 K5 alleles of unrelated individuals with normal skin. The other critical regions of K5 and K14 were unremarkable in this family except for common polymorphisms that have been previously described. The valine at position 7 of the L12 domain is absolutely conserved in all type II keratins, and in other intermediate filament subunits as well, which suggests that this residue makes an important contribution to filament integrity. Secondary structure analysis revealed that alanine at this position markedly reduces both the hydrophobicity and the beta-sheet nature of the L12 domain. This is the first report of a mutation at this position in an intermediate filament subunit and reinforces the importance of this region to filament biology.

Simplifying the molecular mechanisms of human papillomavirus

The human papillomaviruses (HPVs) are associated with several human epithelial diseases. These diseases are confined to cutaneous and mucosal epithelia and comprise papillomas (warts) and benign or malignant neoplasms. Globally, infection by HPVs presents a considerable health problem given that at any one time approximately 10% of the population may have warts of one form or another. Of more serious concern is the prevalence of HPV-associated cervical carcinoma. It is estimated that 500,000 new cases of cervical neoplasia are diagnosed per year (primarily squamous carcinomas). Thus, HPV-associated cancer represents one of the most common cancers afflicting women and is one of the three most common causes of cancer death among women globally.(15) Although some genotypes of human papillomaviruses are clearly associated with the development of cancer (in particular, HPVs 16 and 18) these viruses share significant structural and functional similarity to the nononcogenic genotypes, and one of the puzzles of HPV biology is why essentially similar viruses vary so widely in their oncogenic potential.

Can tissue drug concentrations be monitored by microdialysis during or after isolated limb perfusion for melanoma treatment?

Isolated limb perfusion (ILP) with melphalan is used to treat recurrent melanoma. This study aimed to develop a microdialysis technique for melphalan tissue concentration measurement during ILP. The effects of melphalan concentration (50-600 mu g/ml), microdialysis flow rate (0.55-17.5 mu l/min), probe length (5-50 mm) and temperature (25-41.5 degrees C) on in vitro recovery were studied. In addition, in vivo recovery was measured in rat hindlimbs perfused with melphalan using 50 mm microdialysis probes implanted subcutaneously and into muscle. Both dialysate and tissue sample melphalan concentrations were determined by high performance liquid chromatography. The in vitro recovery of melphalan was not affected by melphalan concentration or temperature, but increased with probe length and decreased with flow rate. The melphalan concentrations in subcutaneous and muscle dialysates were not significantly different. A linear relationship was found between tissue dialysate concentrations and actual tissue concentrations of melphalan (r(2) = 0.97). Microdialysis is a potential method for tissue drug monitoring which may assist in the efficacious use of cytotoxics in human ILP. (C) 2000 Lippincott Williams & Wilkins.

Analysis of Mouse Keratin 6a Regulatory Sequences in Transgenic Mice Reveals Constitutive, Tissue-Specific Expression by a Keratin 6a Minigene

The analysis of keratin 6 expression is complicated by the presence of multiple isoforms that are expressed constitutively in a number of internal stratified epithelia, in palmoplantar epidermis, and in the companion cell layer of the hair follicle. In addition, keratin 6 expression is inducible in interfollicular epidermis and the outer root sheath of the follicle, in response to wounding stimuli, phorbol esters, or retinoic acid. In order to establish the critical regions involved in the regulation of keratin 6a (the dominant isoform in mice), we generated transgenic mice with two different-sized mouse keratin 6a constructs containing either 1.3 kb or 0.12 kb of 5' flanking sequence linked to the lacZ reporter gene. Both constructs also contained the first intron and the 3' flanking sequence of mouse keratin 6a. Ectopic expression of either transgene was not observed. Double-label immunofluorescence analyses demonstrated expression of the reporter gene in keratin 6 expressing tissues, including the hair follicle, tongue, footpad, and nail bed, showing that both transgenes retained keratinocyte-specific expression. Quantitative analysis of beta -galactosidase activity verified that both the 1.3 and 0.12 kb keratin 6a promoter constructs produced similar levels of the reporter. Notably, both constructs were constitutively expressed in the outer root sheath and interfollicular epidermis in the absence of any activating stimulus, suggesting that they lack the regulatory elements that normally silence transcription in these cells. This study has revealed that a keratin 6a minigene contains critical cis elements that mediate tissue-specific expression and that the elements regulating keratin 6 induction lie distal to the 1.3 kb promoter region.

Mathematical models in percutaneous absorption

A number of mathematical models have been used to describe percutaneous absorption kinetics. In general, most of these models have used either diffusion-based or compartmental equations. The object of any mathematical model is to a) be able to represent the processes associated with absorption accurately, b) be able to describe/summarize experimental data with parametric equations or moments, and c) predict kinetics under varying conditions. However, in describing the processes involved, some developed models often suffer from being of too complex a form to be practically useful. In this chapter, we attempt to approach the issue of mathematical modeling in percutaneous absorption from four perspectives. These are to a) describe simple practical models, b) provide an overview of the more complex models, c) summarize some of the more important/useful models used to date, and d) examine sonic practical applications of the models. The range of processes involved in percutaneous absorption and considered in developing the mathematical models in this chapter is shown in Fig. 1. We initially address in vitro skin diffusion models and consider a) constant donor concentration and receptor conditions, b) the corresponding flux, donor, skin, and receptor amount-time profiles for solutions, and c) amount- and flux-time profiles when the donor phase is removed. More complex issues, such as finite-volume donor phase, finite-volume receptor phase, the presence of an efflux. rate constant at the membrane-receptor interphase, and two-layer diffusion, are then considered. We then look at specific models and issues concerned with a) release from topical products, b) use of compartmental models as alternatives to diffusion models, c) concentration-dependent absorption, d) modeling of skin metabolism, e) role of solute-skin-vehicle interactions, f) effects of vehicle loss, a) shunt transport, and h) in vivo diffusion, compartmental, physiological, and deconvolution models. We conclude by examining topics such as a) deep tissue penetration, b) pharmacodynamics, c) iontophoresis, d) sonophoresis, and e) pitfalls in modeling.

Screening for leprosy in immigrants: A decision analysis model

Almost all leprosy cases reported in industrialized countries occur amongst immigrants or refugees from developing countries where leprosy continues to be an important health issue. Screening for leprosy is an important question for governments in countries with immigration and refugee programmes. A decision analysis framework is used to evaluate leprosy screening. The analysis uses a set of criteria and parameters regarding leprosy screening, and available data to estimate the number of cases which would be detected by a leprosy screening programme of immigrants from countries with different leprosy prevalences, compared with a policy of waiting for immigrants who develop symptomatic clinical diseases to present for health care. In a cohort of 100,000 immigrants from high leprosy prevalence regions (3.6/10,000), screening would detect 32 of the 42 cases which would arise in the destination country over the 14 years after migration; from medium prevalence areas (0.7/10,000) 6.3 of the total 8.1 cases would be detected, and from low prevalence regions (0.2/10,600) 1.8 of 2.3 cases. Using Australian data, the migrant mix would produce 74 leprosy cases from 10 years intake; screening would detect 54, and 19 would be diagnosed subsequently after migration. Screening would only produce significant case-yield amongst immigrants from regions or social groups with high leprosy prevalence. Since the number of immigrants to Australia from countries of higher endemnicity is not large routine leprosy screening would have a small impact on case incidence.