Antioxidant and antifungal activities of marrubiin, extracts and essential oil from Marrubium vulgare L. against pathogenic dermatophyte strains.

OBJECTIVE: Medicinal plants extracts and plant-derived compounds are one of the natural sources for discovering new antifungal agents, the objectives of this work were to investigate for the first time the antidermatophytic, antipathogenic activities of methanol, acetone extracts, and essential oil of Marrubium vulgare L. grown in Tunisia and its active compound marrubiin on pathogenic for animals and humans, such as some dermatophytes and pathogenic for plants, and to evaluate antioxidant activities of different extracts with consideration to their chemical compositions. MATERIAL AND METHODS: Acetone and methanol extracts were evaluated by HPLC, the essential oil was also analyzed by GC/MS. PCL assay was used to determine the antioxidant activity. RESULTS: Results showed that methanol and acetone extracts exhibited a significant antioxidant activity (261.41 and 272.90µmol TE/g respectively), while the lowest one was observed in the case of marrubiin and essential oil. The antifungal activity of different extracts, marrubiin and essential oil at two concentrations (20 and 100µg/mL) were screened against the dermatophytes fungi Microsporum gypseum, Microsporum canis, Arthroderma cajetani, Trichophyton mentagrophytes, Trichophyton tonsurans, Epidermophyton floccosum and against two fungi strains (Botrytis cinerea, Pythium ultimum). Among tested extracts, marrubiin at 100µg/mL showed about 50% inhibition for T. mentagrophytes and E. floccosum. The anti-phytopathogenic activity was also carried out, only marrubiin had in activity against B. cinerea at the highest dose (32.40%), while methanol extract of M.vulgare and marrubiin are able to increase the mycelial growth of P. ultimum at the highest concentration (45.15 and 40.30% respectively). CONCLUSION: In our study, we conclude that M.vulgare and marrubiin can be used as natural antioxidants and antifungal agent for treatment of skin dermatophyte infections.

Factors affecting SPF in vitro measurement and correlation with in vivo results.

OBJECTIVE: The in vitro evaluation of SPF is still a problem due to the lack of repeatability and correlation between the in vitro and in vivo data, and many authors are currently working to develop an internationally harmonized method. Very recently, the use of several "adjuvant" ingredients such as boosters, antioxidants, immunomodulators, solvents and film-forming ingredients have further complicated the pattern for product developers that should frequently run in vivo test. The aim of this study was to understand whether a simple and cheap in vitro method could be optimized in order to provide both statistically repeatable and predictive SPF measurement. METHODS: In vitro SPF assessments were carried out on 75 commercial products. The SPF was measured according to two laboratory methods (A and B), using different substrates (PMMA and surgical tape Transpore™), quantity of product and spectrophotometers. In order to evaluate whether a standard technique of spreading could lead to a statistically reliable result, we applied different spreading pressure (100 g and 200 g). Furthermore, we investigate whether other parameters characterizing the product (SPF category, filter and texture) might represent statically significant variables affecting the measures. We then compared the results obtained from in vitro SPF measure of 11 products to in vivo SPF, in order to assess the predictability of in vitro methods. RESULTS: Several problems were encountered in confirming the weakness of the in vitro procedures. Pressure, SPF category, filter and texture did not affect significantly the results. Overall best results were obtained with the B2 method that in terms of repeatability and predictivity provided statistically better results. Method A with Transpore™ tape showed better in vitro-in vivo correlation than Method B with PMMA plates. CONCLUSION: In our investigation, we demonstrated that it is possible for a single laboratory to optimize internal methods and protocols to achieve repeatable and predictive in vitro results, but it is extremely difficult to develop methods reproducible and equally reliable in different laboratories, probably due to "external variables" (e.g. environmental, operator), which are difficult to control.

Fusidic acid betamethasone lipid cream.

Bacterial infections of the skin and soft tissues are frequent disorders. They can be primitive infections (e.g. impetigo, folliculitis) or secondary infections complicating other diseases, particularly atopic dermatitis. The most common aetiologic agent is Staphylococcus aureus. Topical antibiotic therapy may be sufficient in many instances to control these infections. Fusidic acid is an antibiotic used topically on the skin which is very active against S. aureus, including methicillin-resistant strains, and other Gram-positive bacteria. Resistance rates to fusidic acid are stably low. A fusidic acid and betamethasone formulation in a lipid-enriched cream (lipid cream) has been recently developed in order to provide effective antibacterial and anti-inflammatory activities in conjunction with a powerful emollient and moisturising effect. This preparation may be especially useful in patients with atopic-infected eczema.

Phloridzin derivatives inhibiting pro-inflammatory cytokine expression in human cystic fibrosis IB3-1 cells.

Cystic Fibrosis (CF) is the most diffuse autosomal recessive genetic disease affecting Caucasians. A persistent recruitment of neutrophils in the bronchi of CF patients contributes to exacerbate the airway tissue damage, suggesting that modulation of chemokine expression may be an important target for the patient's well being thus the identification of innovative anti-inflammatory drugs is considered a longterm goal to prevent progressive tissue deterioration. Phloridzin, isolated from Malus domestica by a selective molecular imprinting extraction, and its structural analogues, Phloridzin heptapropionate (F1) and Phloridzin tetrapropionate (F2), were initially investigated because of their ability to reduce IL-6 and IL-8 expression in human CF bronchial epithelial cells (IB3-1) stimulated with TNF-α. Release of these cytokines by CF cells was shown to be controlled by the Transcription Factor (TF) NF-kB. The results of the present investigation show that of all the derivatives tested, Phloridzin tetrapropionate (F2) is the most interesting and has greatest potential as it demonstrates inhibitory effects on the expression and production of different cytokines involved in CF inflammation processes, including RANTES, VEGF, GM-CSF, IL-12, G-CSF, MIP-1b, IL-17, IL-10 and IP-10, without any correlated anti-proliferative and pro-apoptotic effects.

The topical vehicle as a key factor in the management of the psoriatic patients' therapy.

This review deals with the importance of the topical vehicle as a key factor in the management of the psoriatic patients' therapy.

[Epidemiological, social and public health aspects of tuberculosis in Ferrara in the 19th century]. / Aspetti epidemiologicie socio-sanitari dellatubercolosi a Ferrara nell'800.

Our first study of tuberculosis in Ferrara during the nineteenth century, whose results have been recently published, focused on disease treatment. Here we present the descriptive analysis of mortality, with the following results being attained: two behavioural patterns are detected with regard to the onset of disease, before and after 1850; TB is a specific disease that affects all parts of the body in all age groups: childhood, and active and passive populations; there are no significant differences with regard to gender; as regards the occupations performed by the deceased, those related to industry and agriculture and to various other activities and services are those with the highest mortality; tuberculosis has a seasonal pattern; summer and autumn are the periods of greatest prevalence (hot weather and humidity are factors that affect the respiratory system); among the forms of tuberculosis it can be observed that up to the year 1850 people died in Ferrara either of pulmonary tuberculosis or TB localised in other areas; from 1851 onward there appears to have been a dramatic change, with a decrease in unspecific diagnosis but the appearance of disease manifestations in its various clinical forms.

Quorum sensing inhibition activity of garlic extract and p-coumaric acid.

AIMS: The goal of this work was to investigate the influence of DMSO, garlic extract and p-coumaric acid on bacterial quorum sensing (QS). METHODS AND RESULTS: The decreases in the QS responses of QS reporter strains Escherichia coli pSB401 and pSB536, Agrobacterium tumefaciens NTL4, Chromobacterium violaceum 5999 and wt 494, Pseudomonas putida IsoF/gfp and environmental Pseudomonas chlororaphis were quantified in relation to growth inhibitory effects. DMSO showed no significant QS-specific effects on the strains tested even at close-to-lethal concentrations. Garlic extracts antagonized the activity of QS receptors LuxR, AhyR and TraR, but were toxic at higher concentrations. P-coumaric acid fully inhibited QS responses of 5999, NTL4 and P. chlororaphis, with no influence on cell viability. CONCLUSIONS: The quorum sensing inhibition activity of garlic was extended to novel receptors, and p-coumaric acid was found to possess previously undescribed QS antagonist properties. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that p-coumaric acid might act as QS inhibitor. Further studies are required to understand its role in the regulation of QS and investigate structurally related compounds.

Design, synthesis and in vivo activity of 9-(S)-dihydroerythromycin derivatives as potent anti-inflammatory agents.

The synthesis of a new class of 9-(S)-dihydroerythromycin derivatives and their anti-inflammatory activity on in vivo PMA assay are described. Modifying the desosamine sugar on the C-3' amino group, it was possible to differentiate between anti-biotic and anti-inflammatory action. The compounds are completely devoid of anti-microbial effects but their anti-inflammatory properties are enhanced. These results strongly suggest the potential of macrolides as a new class of anti-inflammatory agents.

Nanoparticle formulation may affect the stabilization of an antiischemic prodrug.

The prodrug 5'-octanoyl-CPA (Oct-CPA) of the antiischemic N6-cyclopentyladenosine (CPA) has been encapsulated by nanoprecipitation in poly(lactic acid) nanoparticles, which have been recovered by gel-filtration, ultra-centrifugation or dialysis. We have analysed how different surfactants and purification methods can influence the nanoparticle characteristics. The particle sizes have been obtained by scanning electron microscope, whereas a SdFFF system was employed to detect their distributions. The Oct-CPA release from nanoparticles and stabilities in human blood of free and encapsulated prodrug have been analysed by HPLC techniques. The effects of nanoparticles on CPA interaction toward adenosine A1 receptor (its action site) have been analysed using radiolabelled drugs. The smallest nanoparticles and the best degree of homogeneity have been obtained using sodium cholate; the best recovery has been achieved by dialysis, whereas gel-filtration and ultra-centrifugation have induced the greatest removal of surfactants. The release of Oct-CPA was better controlled from the nanoparticles obtained using Pluronic F68 and purified by gel-filtration or ultra-centrifugation. Similarly, these nanoparticles better increased the stability of the prodrug in human blood. In particular, the nanoparticles purified by ultra-centrifugation induced a strong stability to a fraction of the encapsulated Oct-CPA. Any interference by unloaded nanoparticles has been registered for CPA-adenosine A1 receptor interaction.

Vitamin C and 6-amino-vitamin C conjugates of diclofenac: synthesis and evaluation.

Diclofenac (Diclo), its ascorbic acid (AA) or 6-amino-AA (AA-NH2) pro-drugs (AA-Diclo or AA-NH-Diclo) were prepared and evaluated on human retinal pigment epithelium (HRPE) cells to investigate their ability to interact with the vitamin C transporter SVCT2 and their cellular uptake. Furthermore, stabilities in physiological fluids of these compounds were investigated. For kinetic experiments, AA-Diclo was incubated in Tris-HCl buffer, human plasma or whole blood. The extracted samples were analysed by HPLC. AA-Diclo was hydrolysed following first order kinetics in buffer, plasma (t1/2 about 10 h) and whole blood (t1/2 about 3.5 h). Transport and inhibition assays were performed by adding [14C]AA and the above-mentioned unlabelled compounds to plated HRPE cells. Intracellular accumulation was measured incubating HRPE cells with increasing concentrations of unlabelled compounds, following by HPLC analysis. Diclo resulted as a non-competitive inhibitor of AA-transport, showing a Na+-dependent and ascorbate-independent uptake. AA-Diclo behaved as a competitive inhibitor, but it was not transported into cells, whereas its analogue AA-NH-Diclo showed a decreased inhibitory activity. Stability studies suggest AA-Diclo as a potential candidate to enhance the Diclo short half life in vivo. The discovery of a Na+-dependent transporter for Diclo on HRPE cells opens new perspectives for targeting diclofenac into the brain.

Adenosine phosphonoacetic acid is slowly metabolized by NDP kinase.

NDP kinase catalyzes the last step in the phosphorylation of nucleotides. It is also involved in the activation by cellular kinases of nucleoside analogs used in antiviral therapies. Adenosine phosphonoacetic acid, a close analog of ADP already proposed as an inhibitor of ribonucleotide reductase, was found to be a poor substrate for human NDP kinase, as well as a weak inhibitor with an equilibrium dissociation constant of 0.6 mM to be compared to 0.025 mM for ADP. The X-ray structure of a complex of adenosine phosphonoacetic acid and the NDP kinase from Dictyostelium was determined to 2.0 A resolution showing that the analog adopts a binding mode similar to ADP, but that no magnesium ion is present at the active site. As ACP may also interfere with other cellular kinases, its potential as a drug targeting NDP kinase or ribonucleotide reductase is likely to be limited due to strong side effects. The design of new molecules with a narrower specificity and a stronger affinity will benefit from the detailed knowledge of the complex ACP-NDP kinase.

Synthetic approaches to a mononucleotide prodrug of cytarabine.

Synthetic pathways to a mononucleotide prodrug of cytarabine (Ara-C) bearing S-pivaloyl-2-thioethyl (tBuSATE) groups, as biolabile phosphate protections, are reported. Using a common phosphoramidite approach, two different kinds of nucleoside protecting groups have been investigated. During this study, we observed an intermolecular migration of the Boc protecting group in the course of the tert-butyldimethylsilyl ether cleavage using tetrabutyl ammonium fluoride.

Determination of antioxidant efficacy of cosmetic formulations by non-invasive measurements.

BACKGROUND/AIMS: Antioxidants have been proposed, over the last decade, as functional ingredients for anti aging preparations and to prevent and modulate oxidative skin damages. Up to date, beside the photo-induced oxidative skin damages model, none in vivo protocols have shown sufficient reproducibility for the validation of the antioxidant claim for a cosmetic finished product. To this aim, we have recently anticipated a new in vivo protocol based on a microinflammatory model, driven by reactive oxygen species. In the present study our model was validated by comparison with four different instrumental methods. METHODS: The effects of a pre-treatment of two different formulations based on antioxidant functional ingredients, were investigated on forearm skin of 15 healthy volunteers, and compared to a cosmetic base and control area. The instruments considered in the study were Chromameter (CR-300 Minolta), Tewameter TM 210 (Courage-khazaka, Cologne, Germany), Laser Doppler Perfusion Imager (PIM1.0 Lisca Development AB, Sweden), in comparison to DermAnalyzer(R), an easy to use software program developed by us, using the CIE L*a*b* color space parameters. RESULTS: The comparative measurements showed that the antioxidant formulations tested were all able to reduce, in different but statistically significant extent, the intensity of skin redness, and of cutaneous blood flow, when compared to control area (P < 0.0001). CONCLUSIONS: The methyl nicotinate (MN) based microinflammatory model, in conjunction with objective measure- ments, resulted an effective tool for in vivo assessment of oxidative skin injuries. In view of the high level of repeatability, short time of answer and simplicity, the procedure by us developed, is proposed as a possible protocol for the evaluation of in vivo efficacy of antioxidant functional ingredients in cosmetic formulations.

Biliary leaks after laparoscopic cholecystectomy. Results of the endoscopic treatment.

BACKGROUND: Significant postoperative bile leaks occur in approximately 0.8 to 1.1% of patients. The goal of endoscopic therapy is to eliminate the transpapillary pressure gradient, thereby permitting preferential transpapillary bile flow rather than extravasation at the site of leak. METHODS. Sixty-four patients were retrospectively evaluated. Endoscopic treatment comprised endoscopic sphincterotomy followed by insertion of a naso-biliary drainage or a stent. Retained stones were extracted by standard procedures. RESULTS: The cystic duct remnant was the site of bile extravasation in 50 cases, ducts of Luschka were the source in 4 cases, common bile duct in 6 cases and common hepatic duct in 4 cases. Retained bile duct stones were detected in 21 cases and papillary stenosis in 4 cases. Endoscopic therapy involved sphincterotomy in 25 cases with stones extraction in 21 cases followed by nasobiliary drain insertion, and placement of stent in the remainder. Bile leaks resolved in 96.9% of patients, on average 3 days in cases of associated stones or papillary stenosis, and 6.5 days in the remainder. Two cases of mild pancreatitis were evidenced from endoscopic treatment. CONCLUSIONS: Endoscopic management is the treatment of choice for postcholecystectomy bile leaks.

Synthesis and study of 5'-ester prodrugs of N6-cyclopentyladenosine, a selective A1 receptor agonist.

PURPOSE: A series of 5'-esters of N6-cyclopentyladenosine (CPA) were prepared with the aim to improve stability and bioavailability of selective A1 agonists. Log P values, stability, affinity, and activity toward human adenosine A1 receptors were evaluated. METHODS: An appropriate synthetic procedure was adopted to avoid concomitant deamination at position 6. Log P values were obtained by the Mixxor system. The stability of CPA and its 5'-ester was evaluated in human plasma and whole blood and analyzed with high-performance liquid chromatography. The affinities to human A1 receptor expressed by N6-cyclohexyladenosine cells were obtained by binding experiments. The activities were evaluated by measurements of the inhibition of forskolin stimulated 3'-5'-cyclic adenosine monophosphate, performing competitive binding assays. RESULTS: All prodrugs were more lipophilic than CPA, and their hydrolysis, in whole blood and in plasma, was found related, respectively, to the length and hindrance of 5'-substituents. Affinity and activity values indicated a very weak interaction toward adenosine A1 receptor of the intact prodrugs. CONCLUSIONS: We propose 5'-esters of CPA, characterized by suitable lipophilicity and elevated degree of stability in physiological fluids, as possible candidates for CPA prodrugs.

Design, synthesis and enzymatic activity of highly selective human mitochondrial thymidine kinase inhibitors.

Highly selective arabinofuranosyl nucleosides, which inhibit the mitochondrial thymidine kinase (TK-2) without affecting the closely related herpes simplex virus type 1 thymidine kinase (HSV-1 TK), varicella-zoster virus thymidine kinase (VZV-TK), cytosolic thymidine kinase (TK-1) or the multifunctional Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK), have been obtained. SAR studies indicate a close relation between the length of the substituent at the 2' position of the arabinofuranosyl moiety and the inhibitory activity.

2'-O-Acyl/alkyl-substituted arabinosyl nucleosides as inhibitors of human mitochondrial thymidine kinase.

Introduction of a bulky lipophilic acyl entity at the 2'-OH position of both 1-beta-D-arabinofuranosylthymine (araT) and (E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil (BVaraU), consistently resulted in a marked ( approximately 10-fold) increase in the inhibitory activity of these new arabinosyl nucleoside analogues for the mitochondrial thymidine kinase (TK-2)-catalysed conversion of 2 microM [methyl-(3)H]dThd to [methyl-(3)H]dTMP. The most potent derivatives were inhibitory to [methyl-(3)H]dThd phosphorylation by TK-2 within the lower micromolar concentration range. Substitution of the arabinosyl nucleoside derivatives with the acyl groups also dramatically increased the selectivity of these compounds. The inhibitory activity of araT and BVaraU to dThd phosphorylation by other related nucleoside kinases, including herpes simplex virus type 1 TK, varicella-zoster virus TK, and cytosolic TK-1, was completely annihilated upon 2'-O-acyl substitution (IC(50) > or = 1000 microM). Kinetic analysis revealed purely competitive inhibition of 2'-O-acyl-BVaraU against TK-2-catalysed thymidine phosphorylation (K(i)/K(m): 2.3). However, 2'-O-acyl-BVaraU was extremely poorly converted to the corresponding arabinosyl nucleoside 5'-monophosphate by TK-2 as revealed by [gamma-(32)P]phosphate transfer studies from [gamma-(32)P]ATP. Thus, the 2'-O-acyl derivatives of BVaraU did not behave as substrates, but rather as potent and highly selective inhibitors of TK-2. This is the first report on such a highly selective arabinosyl nucleoside inhibitor of mitochondrial TK-2, and opens perspectives for the rational design of selective mitochondrial TK-2 inhibitors.

Novel antioxidant agents deriving from molecular combinations of vitamins C and E analogues: 3,4-dihydroxy-5(R).

Molecular combinations of two antioxidants (i.e., ascorbic acid and the pharmacophore of alpha-tocopherol), namely the 2,3-dihydroxy-2,3-enono-1,4-lactone and the chromane residues, have been designed and tested for their radical scavenging activities. When evaluated for their capability to inhibit malondialdehyde (MDA) production in rat liver microsomal membranes, the 3,4-dihydroxy-5R-2(R,S)-(6-hydroxy-2,5,7,8-tetramethylchroman-2(R,S)yl-methyl)-1,3]dioxolan-4S-yl]-5H-furan-2-one (11a-d), exhibited an interesting activity. In particular the 5R,2R,2R,4S and 5R,2R,2S,4S isomers (11c,d) displayed a potent antioxidant effect compared to the respective synthetic alpha-tocopherol analogue (5) and natural alpha-tocopherol or ascorbic acid, used alone or in combination. Moreover, the mixture of stereoisomers 11a-d also proved to be effective in preventing damage induced by reperfusion on isolated rabbit heart, in particular at the higher concentration of 300 microM. In view of these results our study represents a new approach to potential therapeutic agents for applications in pathological events in which a free radical damage is involved. Design, synthesis and preliminary biological activity are discussed.

Pyrazolo-triazoles as light activable DNA cleaving agents.

In view of the continuous interest in new DNA cleaving compounds, both for the development of new therapeutic agents and for the possible use as reagents in nucleic acids research, a few pyrazolo[3,4-d][1,2,3]triazole derivatives have been obtained and investigated for their antiproliferative activity and capability to cleave DNA, after light-activation. A possible in situ activation, i.e. in neoplastic tissues, of less cytotoxic derivatives, may lead to potential antitumor compounds endowed with high therapeutic indexes.

Design, synthesis and binding at cloned muscarinic receptors of N-[5-(1'-substituted-acetoxymethyl)-3-oxadiazolyl] and N-[4-(1'-substituted-acetoxymethyl)-2-dioxolanyl] dialkyl amines.

Few muscarinic antagonists differentiate between the M4 and M2 muscarinic receptors. In a structure activity study, aimed at discovering leads for the development of a M4 muscarinic receptor-selective antagonist, we have synthesized and tested at cloned muscarinic receptors the binding of a group of dioxolane- or oxadiazole-dialkyl amines, and compared them to our compound 1, which contains the furan nucleus. Although none of these agents were particularly potent at M4 receptors (Kd values were typically 30-70 nM), furan derivatives (-)1 and (+)1 were significantly more potent at M4 receptors than at M2 receptors (approximately 3- and 4-fold, respectively). The dioxolane derivatives 12b and 12c were more than 10-fold selective for the M4 versus the M2 receptors, while the dioxolane derivative 12e was 15-fold more potent at M4 receptors than for M2 receptors. However, these agents bound to M3 receptors with potencies like that for the M4 receptor, so they are not M4-selective. The M4/M2 relative selectivities of some of our compounds are similar to the better hexahydrosiladifenidol derivatives, and may provide some important structural clues for the development of potent and selective M4 antagonists.