Protective role of Portuguese natural mineral waters on skin aging: in vitro evaluation of anti-senescence and anti-oxidant properties.

Natural mineral waters (NMWs) emerge from the earth as springs and their beneficial therapeutic effect has been empirically recognized in different countries. Portugal has diverse NMW resources that are sought for the relief of different afflictions including dermatological complications. However, there is a lack of scientific validation supporting this empiric knowledge. In this study, we aimed to screen the in vitro bioactivity of Portuguese NMWs with different chemical profiles, namely sulfurous/bicarbonate/sodic (SBS), bicarbonate/magnesium, sulfated/calcic, sulfurous/chlorinated/sodic, sulfurous/bicarbonate/fluoridated/sodic, and chlorinated/sodic, focusing on aging-related skin alterations. Mouse skin fibroblasts and macrophages were exposed to culture medium prepared in different NMWs. Cellular viability was evaluated by MTT assay and etoposide-induced senescence was analyzed through the beta-galactosidase staining kit. Wound healing was investigated by the scratch assay, and phototoxicity/photoprotection after UVA irradiation was evaluated using a neutral red solution. ROS production was quantified using the 2'7'-dichlorofluorescin diacetate dye, and the activity of superoxide dismutase (SOD) was analyzed by a commercial kit after lipopolysaccharide exposure. NMWs within the SBS profile demonstrated anti-senescence activity in skin fibroblasts, along with a variable effect on cellular viability. Among the tested NMWs, two decreased cellular senescence and preserved cell viability and were therefore selected for subsequent studies, together with a SBS NMW with therapeutic indications for dermatologic diseases. Overall, the selected NMW promoted wound healing in skin fibroblasts and activated SOD in macrophages, thus suggesting an anti-oxidant effect. None of the NMWs prevented phototoxicity after UV irradiation. Our results shed a light on the anti-aging potential of Portuguese NMW, supporting their putative application in cosmetic or medical products.

Anti-inflammatory potential of Portuguese thermal waters.

In light of Medical Hydrology, thermal waters (TW) are all-natural mineral waters that emerge inside a thermal resort and have therapeutic applications. Their beneficial effect has been empirically recognized for centuries, being indicated for symptom alleviation and/or treatment of several diseases, almost all associated with inflammation. Indeed, an anti-inflammatory effect has been attributed to many different Portuguese TW but there is no scientific validation supporting this empiric knowledge. In the present study, we aimed to investigate the anti-inflammatory properties of 14 TW pertaining to thermal centers located in the Central Region of Portugal, and grouped according to their ionic profile. Mouse macrophage cells stimulated with lipopolysaccharide (LPS), a Toll-like receptor 4 agonist, were exposed to culture medium prepared in TW. Metabolism, nitric oxide (NO) production, inducible nitric oxide synthase (iNOS) expression levels and the scavenging capacity of TW, were investigated in vitro. 11 out of 14 TW reduced NO production and/or iNOS expression, and/or scavenging activity, in macrophages exposed to LPS. The sulphated/calcic TW did not show any effect on at least one of the inflammatory parameters evaluated. Two sulphurous/bicarbonate/sodic TW and the sulphurous/chlorinated/sodic TW promoted an increase in NO production and/or iNOS expression. Our results validate, for the first time, the anti-inflammatory properties of Portuguese TW, supporting their therapeutic use in the treatment of inflammation-related diseases and promoting their putative application in cosmetic products and medical devices.

Bacteriocin production of the probiotic Lactobacillus acidophilus KS400.

In the last years, the use of probiotics, including Lactobacillus species, has received much attention to prevent and treat vaginal disorders. These species have been described as having the ability to colonize the epithelial surface and produce antimicrobial metabolites that are able to control the remaining vaginal microflora. This study aimed to identify and characterize, for the first time, a bacteriocin natively produced by Lactobacillus acidophilus KS400 (probiotic strain from Gynoflor®-Medinova AG, Switzerland) and its antimicrobial activity against relevant urogenital pathogens. After organic acids and hydrogen peroxide neutralization in the fermented Lactobacillus acidophilus KS400 culture medium, bacteriocin activity was tested against the indicator microorganism Lactobacillus delbrueckii ATCC9649. The fermentation of Lactobacillus acidophilus KS400 for bacteriocin production was carried out in batch mode, and its antimicrobial activity, optical density and pH were monitored. After production and extraction, the bacteriocin molecular weight was estimated by electrophoresis and tested against vaginal pathogenic microorganisms. As described for other bacteriocins, batch fermentation profiles indicated that bacteriocin production occurs during the exponential growth phase of the lactobacilli, and declines during their stationary growth phase. The molecular weight of the bacteriocin is approximately 7.5 kDa. The bacteriocin containing protein extract was shown to inhibit the growth of Gardnerella vaginalis, Streptococcus agalactiae, Pseudomonas aeruginosa and the indicator strain Lactobacillus delbrueckii ATCC9649. We conclude that L. acidophilus KS400 produces bacteriocin with antimicrobial activity against relevant urogenital pathogens.

Microbiological quality control of non-sterile compounded medicines prepared in a Portuguese hospital centre.

OBJECTIVES: This work aimed to evaluate the quality of non-sterile formulations compounded at Centro Hospitalar Cova da Beira (Covilhã, Portugal) immediately after preparation and up to the defined 'beyond-use date'. METHODS: Microbiological quality control tests were performed in accordance with monograph 5.1.4 of the European Pharmacopoeia 8.0. Samples of compounded products were collected from January to December 2014 after preparation and were analysed immediately and reanalysed after storage under the established conditions, for each preparation. RESULTS: In the test period, 392 preparations were analysed, corresponding to 24 different formulations (8 intermediate preparations, 11 oral solutions/suspensions and 5 topical preparations). All preparations were in accordance with the pharmacopoeia specifications immediately after preparation. However, for the formulations 'prednisolone oral solution (5 mg/mL)' and 'nitroglycerine and cinchocaine ointment (0.25%/0.5%)', the microbial counts of some batches exceeded the defined limits after storage up to the beyond-use date. CONCLUSIONS: These results show that the compounding practices implemented at this pharmacy department are able to ensure the microbiological quality of compounded products. This microbiological quality control methodology also allowed identification of the need to replace formulations shown not to be stable throughout the storage period. On the basis of these results, a monthly routine of microbiological control of a random sample of compounded medicines was established in order to ensure their quality and safety for use.

Are plant extracts a potential therapeutic approach for genital infections?

More than 40% of the new drugs registered from 1981 to 2006 were obtained, derived or inspired from natural compounds. The influence of natural products in the anti-infective area is quite marked, being a great percentage of drugs derived or extracted from natural products. Vaginal infections are one of the most common reasons a women visits a gynecologist. Given the high popularity of natural therapies among women who suffer from chronic infections, it is urgent for women's healthcare providers to be knowledgeable about such therapies. Additionally, many phytotherapeutic products have been suggested as natural sources of antimicrobial compounds. The increased resistance to conventional antibiotics is one of the main factors justifying the search and development of new antimicrobial agents, especially those of natural origin. Currently, phytochemicals are considered by the scientific community as very attractive targets for potential drug discovery and therapy. In this review, we will focus on the most relevant reports published during the last twenty years about the antimicrobial activity of plant extracts upon microorganisms most frequently involved in genital infections, such as Candida spp., Gardnerella vaginalis, Trichomonas vaginalis and Human papillomavirus. The relationship between their composition and the antimicrobial effects will be highlighted and vaginal therapeutic delivery systems that vehicle plant extracts both commercialized and under investigation will be included.

In vitro assessment of gentian violet anti- candida activity.

BACKGROUND/AIM: Recurrent vulvovaginal candidosis (RVVC) needs alternative therapeutic approaches. Gentian violet (GeV) has been traditionally used to treat mucocutaneous candidosis. The aim of the present study was to evaluate the in vitro activity of GeV against Candida spp. and contribute to clarify the mechanism of action, supporting its clinical therapeutic use. METHODS: Seventeen clinical Candida isolates from RVVC and one C. albicans type collection (ATCC 10231) were studied; the antifungal activity of GeV was evaluated according to the CLSI M27-A3 protocol. To elucidate its mechanism of action, cells were stained with propidium iodide and afterwards analyzed by flow cytometer. RESULTS: GeV showed a fungicidal activity against most Candida spp. C. albicans and C. tropicalis were the most susceptible species. Minimal lethal concentrations were similar to minimal inhibitory concentrations for most tested strains. The fungicidal effect was not related to a primary lesion of the cytoplasmic membrane. CONCLUSION: In accordance with our findings, GeV is a valuable potent fungicidal drug to be used topically, isolated or in combination with oral antifungal drugs, particularly in RVVC cases.

The anti-Candida activity of Thymbra capitata essential oil: effect upon pre-formed biofilm.

ETHNOPHARMACOLOGICAL RELEVANCE: [corrected] Thymbra capitata essential oil is traditionally considered to exhibit powerful antiseptic properties, thus being used to treat cutaneous infections. The aim of the present study was to evaluate the effect of Thymbra capitata essential oil upon pre-formed biofilm of different Candida strains while comparing it with the activity against planktonic cells. MATERIALS AND METHODS: Fifteen Candida isolates were included, corresponding to clinical and collection type strains. Essential oil was obtained by hydrodistillation and its composition analysed by GC/MS. Activity upon planktonic cells was evaluated according to M27-A3 macromethod. Its effect upon 24h preformed biofilm biomass was determined using the crystal violet procedure and the metabolic activity was studied applying the XTT/menadione technique. RESULTS: Biofilm biomass and metabolic activity of all tested species were reduced up to 50% at MIC values. The effect was more pronounced at double MIC values, achieving >80% reduction, except for Candida albicans that presented a more resistant profile (62%). CONCLUSION: Thymbra capitata essential oil presented an important effect upon Candida biofilms. It is proposed as a valuable antifungal product to be used in an appropriate pharmaceutical formulation for the management of resistant mucocutaneous candidosis.

The relationship between Candida species charge density and chitosan activity evaluated by ion-exchange chromatography.

Chitosan, a natural biopolymer presents antifungal activity that seems to be dependent on the interaction of its cationic amino groups and yeast cell surface. In this work we used ion-exchange chromatography to assess the surface charge density of Candida species and subsequently to relate this with their sensitivity profile to chitosan. The ability of several strains from distinct Candida species to interact with strong anionic and cationic exchangers was tested and the yeasts charge surface was assessed by measuring the zeta potential. Our results showed that all the yeast cells tested presented no interaction with the cationic resin and a species-related pattern of interaction was observed with the anionic resin. Specifically, regarding the Q-Sepharose support, Candida glabrata showed the lower retention affinity, followed by Candida albicans, presenting Candida tropicalis an intermediate profile; Candida parapsilosis and Candida guilliermondii revealed a stronger ionic interaction. The yeasts retention synergy in the anionic resin corroborates with the zeta potential outcomes. The behavior observed fit with sensitivity patterns to chitosan as the most susceptible species to chitosan presented higher affinity to the anionic resin in contrast to the less sensitive ones (C. albicans and C. glabrata). This data confirms and reinforces that chitosan activity is probably mediated by an ionic reaction between its amino free groups and ionic charges at the cell surface.

Sodium Tripolyphosphate: an excipient with intrinsic in vitro anti-Candida activity.

Sodium Tripolyphosphate (STPP) is a food additive that is being used in the development of micro and nanoparticles as it induces ionic interactions with chitosan molecules. Although the ability of STPP to inhibit the growth of several food contaminants has been reported, studies on its activity against clinical isolates are scarce. Candida spp. are common causative agents of mucocutaneous infections including the vulvovaginal tegument and new therapeutic approaches are needed in order to treat resistant and recurrent cases. The aim of this study was to evaluate in vitro both antifungal (anti-Candida spp.) activity, and cytotoxicity, on human dermal fibroblasts, of STPP solutions. STPP showed an inhibitory species-dependent activity against several Candida spp. strains being particularly active on C. glabrata, followed by C. guilliermondii. In vitro, STPP showed a concentration dependent cytotoxicity. Therefore STPP use, in low concentrations, seems to be interesting in the development of drug delivery systems for the treatment of vulvovaginal candidosis.

Anti-Candida activity of a chitosan hydrogel: mechanism of action and cytotoxicity profile.

Candida spp. are common causative agents of mucocutaneous infections. New therapeutic antifungal drugs are needed to treat chronic disease as these are frequently clinically resistant to azols. Chitosan, among other possible vehicles for active compounds, shows an added value as it appears to have intrinsic antimicrobial properties. The aim of the present study was to evaluate the anti-Candida activity of a medium-molecular-weight chitosan hydrogel (CH), to clarify its possible mechanism of action and to evaluate its cytotoxicity on human fibroblasts. CH antifungal activity was assessed according to CLSI reference M27-A3 protocol; its mechanism of action was investigated by flow cytometry, and its cytotoxicity was studied by MTT assay. CH demonstrated a full inhibition of C. tropicalis, C. krusei, C. guilliermondii and C. parapsilosis growth while impairing C. albicans and C. glabrata viability. Flow cytometry tests showed that CH acts by inducing primary lesion of the cytoplasmic membrane. However, CH showed no cytotoxic effect upon human fibroblasts cells. Resistant strains will require new therapeutic approaches. Chitosan being a good carrier and having itself anti-Candida activity seems to be a promising vehicle to be used for the treatment of mucocutaneous candidosis.

Anti-Candida activity of essential oils.

Anti-Candida activity of essential oils has been widely studied and as a consequence they are being investigated as possible alternatives or complementary therapeutic agents for candidosis. We reviewed the most studied essential oils concerning chemical composition and in vitro/in vivo studies under the perspective of their possible clinical use.