Diverse Effects of Natural and Synthetic Surfactants on the Inhibition of Staphylococcus aureus Biofilm.

A major challenge in the biomedical field is the creation of materials and coating strategies that effectively limit the onset of biofilm-associated infections on medical devices. Biosurfactants are well known and appreciated for their antimicrobial/anti-adhesive/anti-biofilm properties, low toxicity, and biocompatibility. In this study, the rhamnolipid produced by Pseudomonas aeruginosa 89 (R89BS) was characterized by HPLC-MS/MS and its ability to modify cell surface hydrophobicity and membrane permeability as well as its antimicrobial, anti-adhesive, and anti-biofilm activity against Staphylococcus aureus were compared to two commonly used surfactants of synthetic origin: Tween® 80 and TritonTM X-100. The R89BS crude extract showed a grade of purity of 91.4% and was composed by 70.6% of mono-rhamnolipids and 20.8% of di-rhamnolipids. The biological activities of R89BS towards S. aureus were higher than those of the two synthetic surfactants. In particular, the anti-adhesive and anti-biofilm properties of R89BS and of its purified mono- and di-congeners were similar. R89BS inhibition of S. aureus adhesion and biofilm formation was ~97% and 85%, respectively, and resulted in an increased inhibition of about 33% after 6 h and of about 39% after 72 h when compared to their chemical counterparts. These results suggest a possible applicability of R89BS as a protective coating agent to limit implant colonization.

Δ9-cis-Tetrahydrocannabinol: Natural Occurrence, Chirality, and Pharmacology.

The cis-stereoisomers of Δ9-THC [(-)-3 and (+)-3] were identified and quantified in a series of low-THC-containing varieties of Cannabis sativa registered in Europe as fiber hemp and in research accessions of cannabis. While Δ9-cis-THC (3) occurs in cannabis fiber hemp in the concentration range of (-)-Δ9-trans-THC [(-)-1], it was undetectable in a sample of high-THC-containing medicinal cannabis. Natural Δ9-cis-THC (3) is scalemic (ca. 80-90% enantiomeric purity), and the absolute configuration of the major enantiomer was established as 6aS,10aR [(-)-3] by chiral chromatographic comparison with a sample available by asymmetric synthesis. The major enantiomer, (-)-Δ9-cis-THC [(-)-3], was characterized as a partial cannabinoid agonist in vitro and elicited a full tetrad response in mice at 50 mg/kg doses. The current legal discrimination between narcotic and non-narcotic cannabis varieties centers on the contents of "Δ9-THC and isomers" and needs therefore revision, or at least a more specific wording, to account for the presence of Δ9-cis-THCs [(+)-3 and (-)-3] in cannabis fiber hemp varieties.

Medical-Grade Silicone Coated with Rhamnolipid R89 Is Effective against Staphylococcus spp. Biofilms.

Staphylococcus aureus and Staphylococcus epidermidis are considered two of the most important pathogens, and their biofilms frequently cause device-associated infections. Microbial biosurfactants recently emerged as a new generation of anti-adhesive and anti-biofilm agents for coating implantable devices to preserve biocompatibility. In this study, R89 biosurfactant (R89BS) was evaluated as an anti-biofilm coating on medical-grade silicone. R89BS is composed of homologues of the mono- (75%) and di-rhamnolipid (25%) families, as evidenced by mass spectrometry analysis. The antimicrobial activity against Staphylococcus spp. planktonic and sessile cells was evaluated by microdilution and metabolic activity assays. R89BS inhibited S. aureus and S. epidermidis growth with minimal inhibitory concentrations (MIC99) of 0.06 and 0.12 mg/mL, respectively and dispersed their pre-formed biofilms up to 93%. Silicone elastomeric discs (SEDs) coated by R89BS simple adsorption significantly counteracted Staphylococcus spp. biofilm formation, in terms of both built-up biomass (up to 60% inhibition at 72 h) and cell metabolic activity (up to 68% inhibition at 72 h). SEM analysis revealed significant inhibition of the amount of biofilm-covered surface. No cytotoxic effect on eukaryotic cells was detected at concentrations up to 0.2 mg/mL. R89BS-coated SEDs satisfy biocompatibility requirements for leaching products. Results indicate that rhamnolipid coatings are effective anti-biofilm treatments and represent a promising strategy for the prevention of infection associated with implantable devices.

O-Methyl Phytocannabinoids: Semi-synthesis, Analysis in Cannabis Flowerheads, and Biological Activity.

A general protocol for the selective mono-O-methylation of resorcinyl phytocannabinoids was developed. The availability of semisynthetic monomethyl analogues of cannabigerol, cannabidiol, and cannabidivarin (1A: -3A: , respectively) made it possible to quantify these minor phytocannabinoids in about 40 different chemotypes of fiber hemp. No chemotype significantly accumulated mono-O-methyl cannabidiol (2B: ) or its lower homologue (3B: ), while at least three chemotypes containing consistent amounts (≥ 400 mg/kg) of O-methylcannabigerol (1B: ) were identified. O-Methylation of alkyl phytocannabinoids (1B: -3B: ) does not significantly change the activity on peroxisome proliferator-activated receptors in contrast to what was reported for phenethyl analogues.

The Bibenzyl Canniprene Inhibits the Production of Pro-Inflammatory Eicosanoids and Selectively Accumulates in Some Cannabis sativa Strains.

Canniprene (1), an isoprenylated bibenzyl unique to Cannabis sativa, can be vaporized and therefore potentially inhaled from marijuana. Canniprene (1) potently inhibited the production of inflammatory eicosanoids via the 5-lipoxygenase pathway (IC50 0.4 µM) and also affected the generation of prostaglandins via the cyclooxygenase/microsomal prostaglandin E2 synthase pathway (IC50 10 µM), while the related spiranoid bibenzyls cannabispiranol (2) and cannabispirenone (3) were almost inactive in these bioassays. The concentration of canniprene (1) was investigated in the leaves of 160 strains of C. sativa, showing wide variations, from traces to >0.2%, but no correlation was found between its accumulation and a specific phytocannabinoid profile.

Lipopeptides from Bacillus subtilis AC7 inhibit adhesion and biofilm formation of Candida albicans on silicone.

Candida albicans is the major fungus that colonises medical implants, causing device-associated infections with high mortality. Antagonistic bacterial products with interesting biological properties, such as biosurfactants, have recently been considered for biofilm prevention. This study investigated the activity of lipopeptide biosurfactant produced by Bacillus subtilis AC7 (AC7 BS) against adhesion and biofilm formation of C. albicans on medical-grade silicone elastomeric disks (SEDs). Chemical analysis, stability, surface activities of AC7 BS crude extract and physicochemical characterisation of the coated silicone disk surfaces were also carried out. AC7 BS showed a good reduction of water surface tension, low critical micelle concentration, good emulsification activity, thermal resistance and pH stability. Co-incubation with 2 mg ml(-1) AC7 BS significantly reduced adhesion and biofilm formation of three C. albicans strains on SEDs in a range of 67-69 % and of 56-57 %, respectively. On pre-coated SEDs, fungal adhesion and biofilm formation were reduced by 57-62 % and 46-47 %, respectively. Additionally, AC7 BS did not inhibit viability of C. albicans strains in both planktonic and sessile form. Chemical analysis of the crude extract revealed the presence of two families of lipopeptides, principally surfactin and a lower percentage of fengycin. The evaluation of surface wettability indicated that AC7 BS coating of SEDs surface was successful although uneven. AC7 BS significantly prohibits the initial deposition of C. albicans and slows biofilm growth, suggesting a potential role of biosurfactant coatings for preventing fungal infection associated with silicone medical devices.

The high-resolution crystal structure of periplasmic Haemophilus influenzae NAD nucleotidase reveals a novel enzymatic function of human CD73 related to NAD metabolism.

Haemophilus influenzae is a major pathogen of the respiratory tract in humans that has developed the capability to exploit host NAD(P) for its nicotinamide dinucleotide requirement. This strategy is organized around a periplasmic enzyme termed NadN (NAD nucleotidase), which plays a central role by degrading NAD into adenosine and NR (nicotinamide riboside), the latter being subsequently internalized by a specific permease. We performed a biochemical and structural investigation on H. influenzae NadN which determined that the enzyme is a Zn2+-dependent 5'-nucleotidase also endowed with NAD(P) pyrophosphatase activity. A 1.3 Å resolution structural analysis revealed a remarkable conformational change that occurs during catalysis between the open and closed forms of the enzyme. NadN showed a broad substrate specificity, recognizing either mono- or di-nucleotide nicotinamides and different adenosine phosphates with a maximal activity on 5'-adenosine monophosphate. Sequence and structural analysis of H. influenzae NadN led us to discover that human CD73 is capable of processing both NAD and NMN, therefore disclosing a possible novel function of human CD73 in systemic NAD metabolism. Our data may prove to be useful for inhibitor design and disclosed unanticipated fascinating evolutionary relationships.

LC/ESI-MS/MS characterisation of lipopeptide biosurfactants produced by the Bacillus licheniformis V9T14 strain.

Lipopeptide biosurfactants produced by the Bacillus licheniformis V9T14 strain showed an interesting anti-adhesion activity against biofilm formation of human pathogenic bacterial strains. The chemical characterisation of the crude extract of V9T14 strain was first developed through electrospray ionisation mass spectrometry (ESI-MS) and ESI-MS/MS direct infusions: two sets of molecular ion species belonging to the fengycin and surfactin families were revealed and their structures defined, interpreting their product ion spectra. The LC/ESI-MS analysis of the crude extract allowed to separate in different chromatogram ranges the homologues and the isoforms of the two lipopeptide families. The extract was then fractionated by silica gel chromatography in two main fractions, I and II. The purified biosurfactants were analysed through a new, rapid and suitable LC/ESI-MS/MS method, which allowed characterising the composition and the structures of the produced lipopeptides. LC/ESI-MS/MS analysis of fraction I showed the presence of C(13), C(14) and C(15) surfactin homologues, whose structures were confirmed by the product ion spectra of the sodiated molecules [M + Na](+) at m/z 1030, 1044 and 1058. LC/ESI-MS/MS analysis of fraction II confirmed the presence of two main fengycin isoforms, with the protonated molecules [M + H](+) at m/z 1478 and 1506 corresponding to C(17) fengycin A and C(17) fengycin B, respectively. Other homologues (C(14) to C(16)) were revealed and confirmed as belonging to fengycin A or B according to the retention times and the product ions generated, although with the same nominal mass. Finally, a relative percentage content of each homologue for both lipopeptides families in the whole extract was proposed.

Pattern recognition and genetic algorithms for discrimination of orange juices and reduction of significant components from headspace solid-phase microextraction.

INTRODUCTION: Orange (Citrus sinensis L.) juice comprises a complex mixture of volatile components that are difficult to identify and quantify. Classification and discrimination of the varieties on the basis of the volatile composition could help to guarantee the quality of a juice and to detect possible adulteration of the product. OBJECTIVE: To provide information on the amounts of volatile constituents in fresh-squeezed juices from four orange cultivars and to establish suitable discrimination rules to differentiate orange juices using new chemometric approaches. METHODOLOGY: Fresh juices of four orange cultivars were analysed by headspace solid-phase microextraction (HS-SPME) coupled with GC-MS. Principal component analysis, linear discriminant analysis and heuristic methods, such as neural networks, allowed clustering of the data from HS-SPME analysis while genetic algorithms addressed the problem of data reduction. To check the quality of the results the chemometric techniques were also evaluated on a sample. RESULTS: Thirty volatile compounds were identified by HS-SPME and GC-MS analyses and their relative amounts calculated. Differences in composition of orange juice volatile components were observed. The chosen orange cultivars could be discriminated using neural networks, genetic relocation algorithms and linear discriminant analysis. Genetic algorithms applied to the data were also able to detect the most significant compounds. CONCLUSIONS: SPME is a useful technique to investigate orange juice volatile composition and a flexible chemometric approach is able to correctly separate the juices.

Development and validation of a solid-phase extraction and gas chromatography-tandem mass spectrometry method for the determination of isopropyl-9H-thioxanthen-9-one in carton packaged milk.

A simple and efficient method for the determination of isopropyl-9H-thioxanten-9-one (ITX) in different fat content milk samples and baby milk samples stored in packaged cartons was developed and validated. Samples were extracted using solid-phase extraction (SPE) and analysed by gas chromatography-tandem mass spectrometry operated in selected reaction monitoring mode (SRM). Validation was carried out in terms of limit of detection (LOD), limit of quantitation (LOQ), linearity, precision and trueness. LOD and LOQ values in the low microg/L were achieved, whereas linearity was established within 0.5-500 microg/L range. Good precision was obtained both in terms of intra-day repeatability and inter-day precision on two concentration levels (RSD% lower than 2%). Good percentage recoveries were obtained (92.0-102.0%) even in the presence of high amount of fat. Finally, the developed method was successfully applied to analyse a number of commercial milk samples with different fat content and baby milk samples.

Comparison of volatile concentrations in hand-squeezed juices of four different lemon varieties.

Fresh juices of four Italian cultivars of lemons (Citrus limon Burm) have been analyzed by headspace solid phase microextraction coupled to gas chromatography and gas chromatography-mass spectrometry (GC-MS). The best results have been obtained with the 2 cm 50/30 microm divinylbenzene/carboxen on poly(dimethylsiloxane) fiber, using a homogenization time of 1 h at 40 degrees C and a sampling period of 30 min. A total of 35 volatile compounds have been identified by GC-MS, and their relative amounts have been calculated by adding internal standard to the samples. Differences in composition of lemon juices volatile components have been observed. Verdello Siracusano lemon juice has the highest amount of volatile compounds (50.28 mg/L), followed by Interdonato (8.39 mg/L), Primo Fiore Capo d'Orlando (5.75 mg/L), and Femminello Siracusano (2.62 mg/L) juices. Volatile compounds mainly consist of mono- and sesquiterpene hydrocarbons and oxygenated molecules (aldheydes, monoterpene alcohols, and monoterpene esters). Headspace solid phase microextraction coupled to a gas chromatograph equipped with a specific sulfur detector, a sulfur chemiluminescence detector, let us detect and quantify dimethyl sulfide compound at the microgram/liter level in lemon juices.

Simultaneous, stability indicating, HPLC-DAD determination of guaifenesin and methyl and propyl-parabens in cough syrup.

A stability indicating high performance liquid chromatography procedure has been developed for the simultaneous determination of guaifenesin (GUA), methyl p-hydroxybenzoate (MHB) and propyl p-hydroxybenzoate (PHB) in a commercial cough syrup dosage form. The method was specific and stability indicating as chromatographic conditions were selected to provide adequate separation of GUA, MHB and PHB from the putative degradation products guaiacol (GUAI) and p-hydroxybenzoic acid (HBA) as well as from excipients. The isocratic separation and quantitation were achieved within 17 min on a 150-mm column with an ether-linked phenyl stationary phase and a hydrophilic endcapping. The mobile phase was constituted of eluant A: aqueous phosphate buffer (pH 3.0, 10 mM)/acetonitrile 25/75 (v/v) and eluant B:methanol; the A:B ratio was 85:15 (v/v) with a flow rate 1 ml min-1 and detection of analytes at 254 and 276 nm. The method showed good linearity for the GUA-MHB-PHB mixture in the 95-285, 4-12, and 1-3 microg ml-1 ranges, respectively, being all the square of the correlation coefficients greater than 0.999. The interday R.S.D.s were 1.17, 1.14, and 0.91%, for GUA, MHB, and PHP, respectively. The method demonstrated also to be accurate; indeed the average recoveries, at 100% of the target assay concentration, were 100.5, 100.3, and 100.7% with relative standard deviations of 0.8, 0.7, and 0.4% for GUA, MHB, and PHB, respectively, from laboratory prepared samples. The applicability of the method was evaluated in commercial dosage form analysis as well as in stability studies.

Impurity analysis of retinoic acid samples.

The structure of an impurity contained in samples of all trans-retinoic acid was established by means of NMR and MS spectra, and confirmed by X-ray diffraction analysis. The chemical structure of the impurity 2 was found to be strictly correlated to the synthetic procedure employed for the preparation of the retinoic acid samples. Single crystal analysis allowed us to characterise the molecular conformation and the crystal structure of 2.

Synthesis and biological activity of mustard derivatives of combretastatins.

A series of chimeric compounds bearing the combretastatin and the nitrogen mustard cores were synthesized. All the compounds were cytotoxic and inhibited tubulin polymerization. When combretastatin was joined to chlorambucil via an ester linkage, the resultant compound proved to be significantly more potent than the two compounds put together. When combretastatin was joined to nitrogen mustard via an ether linkage or when a true hybrid was synthesized, loss of potency was observed. Nonetheless, these latter compounds appeared to be more efficacious and surprisingly were able to inhibit tubulin depolymerization at high concentrations.

LC-ESI-MS/MS characterization of strophanthin-K.

A liquid chromatography-mass spectrometry (LC-MS) method was developed for the characterization of strophanthin-K, a mixture of cardiac glycosides extracted from the seeds of Strophanthus kombe. The method is based on the separation of the cardenolides using high performance liquid chromatography (HPLC) followed by detection with electrospray ionization mass-spectrometry (ESI-MS). Chromatographic separation of the analytes was achieved on a RP C-18 column using water: 1% formic acid in water (v/v):acetonitrile gradient mobile phase. Strophanthin-K glycosides studied in ESI-MS in negative ion mode formed abundant adduct ions [M+HCOO](-) while the pseudomolecular ions [M-H](-) are obtained in ESI-MS/MS experiments. Six different cardiac glycosides were identified and characterized: k-strophanthoside, k-strophanthin-beta, helveticoside (erysimin), erysimoside, cymarin and neoglucoerysimoside. Forced degradation investigations done with strophanthin-K showed that k-strophanthidin (the aglycone of strophanthin-K glycosides) was the main product of degradation in acidic conditions; however, in basic conditions, the hydrolysis of the unsaturated 17beta-lactones to the corresponding gamma-hydroxy acids was the predominant degradation pathway.