Fabrizio Gelmini

GC–MS profiling of the phytochemical constituents of the oleoresin from Copaifera langsdorffii Desf. and a preliminary in vivo evaluation of its antipsoriatic effect

Copaiba is the oleoresin (OR) obtained from Copaifera (Fabaceae), a neotropical tree which grows in Amazon regions. The balsam, constituted by an essential oil and a resinous fraction is used as folkloristic remedy in the treatment of several inflammatory diseases and for its antioxidant and antibacterial properties. Aim of this work was (a) to carry out a characterization by GC-MS of the volatile and nonvolatile constituents of Copaifera langsdorffii Desf. oleoresin (OR); (b) to investigate the mechanism of its anti-inflammatory activity; (c) to evaluate its antipsoriatic effect after oral intake/topical application. The volatile fraction (yield: 22.51%, w/w) shows: α-bergamotene (48.38%), α-himachalene (11.17%), β-selinene (5.00%) and β-caryophyllene (5.47%). The OR residue (77.49%, w/w), after derivatization, showed as main constituents the following compounds: copalic, abietic, daniellic, lambertinic, labd-7-en-15-oic, pimaric, isopimaric acids and kaur16-en18-oic acid. Preincubation of LPS-stimulated human THP-1 monocytes with increasing concentrations of the OR purified fraction (OR-PF), containing diterpene acids, diterpenes and sesquiterpenes, reduced the release of pro-inflammatory cytokines (IL-1β, IL-6, TNFα) in a dose-range of 0.1-10 μM. In addition, in cell culture system of human THP-1 monocytes, 1 μM OR-PF counteracts LPS-driven NF-κB nuclear translocation. In a preliminary clinical trial three patients affected by chronic psoriasis, treated with oral intake or topical application of the OR, exhibited a significant improvement of the typical signs of this disease, i.e. erythema, skin thickness, and scaliness. In conclusion, the results of this work, beside an extensive analytical characterization of the OR chemical composition, provide strong evidences that its anti-inflammatory activity is related to the inhibition of the NF-κB nuclear translocation, and consequently of proinflammatory cytokines secretion.

Preservation of Endothelium Nitric Oxide Release by Pulsatile Flow Cardiopulmonary Bypass When Compared With Continuous Flow

The aim of this work is to analyze endothelium nitric oxide (NO) release in patients undergoing continuous or pulsatile flow cardiopulmonary bypass (CPB). Nine patients operated under continuous flow CPB, and nine patients on pulsatile flow CPB were enrolled. Plasma samples were withdrawn for the chemiluminescence detection of nitrite and nitrate. Moreover the cellular component was withdrawn for the detection of nitric oxide synthase (NOS) activity in the erythrocytes, and an estimation of systemic inflammatory response was carried out. Significant reduction in the intraoperative concentration with respect to the preoperative was observed only under continuous flow CPB for both nitrite and NO(x) (nitrite + nitrate) concentration (P = 0.010 and P = 0.016, respectively). Significant difference in intraoperative nitrite concentration was also observed between the groups (P = 0.012). Finally, erythrocytes showed a certain endothelial NOS activity, which did not differ between the groups, and no differences in the inflammatory response were pointed out. The significant reduction of NO(2)(-) concentration under continuous perfusion revealed the strong connection among perfusion modality, endothelial NO release, and plasmatic nitrite concentration. The similar erythrocyte eNOS activity between the groups revealed that the differences in blood NO metabolites are mainly ascribable to the endothelium release.

An analytical and theoretical approach for the profiling of the antioxidant activity of essential oils: the case of Rosmarinus officinalis L.

The antioxidant constituents of essential oils (EOs) of Rosmarinus officinalis L. (α-pinene chemotype) were isolated at the flowering (A), post-flowering (B), and vegetative stages (C). GC-MS was used to analyze total chemical composition, Folin-Ciocalteau and Prussian blue methods for reducing substances. Radical scavenging capacity (DPPH test, IC(50) 36.78±0.38, 79.69±1.54, 111.94±2.56μL) and anti-lipoperoxidant activity (TBARS, IC(50) 0.42±0.04, 1.20±0.06μL, 4.07±0.05μL) differed widely in the three stages. Antioxidant activity, identified after silica gel fractionation chromatography, was closely related (R(2)=0.9959) to each EOs content of hydroxilated derivatives, (containing alcohols, phenols and 1,8 cineole): 15.26±0.12%, 7.22±0.06%, and 5.09±0.10% in EOs from A, B, and C. Modeling the C, H, O terpenes in a simulated phospholipid bilayer indicated that anti-lipoperoxidant activity depended on the stability and rapidity of their interactions with the membrane bilayer components, and their positioning over its surface.

Profile of nitric oxide (NO) metabolites (nitrate, nitrite and N-nitroso groups) in honeys of different botanical origins: nitrate accumulation as index of origin, quality and of therapeutic opportunities.

Besides dermoprotective activity, honey also has a strong gastroprotective effect, from salivary reduction of nitrate (NO(3)(-)) to nitrite (NO(2)(-)) and intragastric formation of nitric oxide (NO), this lastly involved in the preservation of the gastric mucosa capillaries and in boosting mucous production. Aim of this work is to profile the distribution of NO metabolites (NO(3)(-), NO(2)(-) and total N-nitroso-groups, N-NO) in a set of honeys (n=54) of different botanical origins, using a chemiluminescence based technique (NO-analyzer, NOA). All the honeys contained appreciable amounts of NO(3)(-) (from 1.63+/-0.04 to 482.98+/-5.34 mg/kg), the highest in honeydew honeys (10-40 times than in nectar honeys). Low levels of NO(2)(-) were found in all samples (0.01+/-0.00 to 0.56+/-0.01 mg/kg). N-NO groups, at trace levels in some nectar honeys, were higher in honeydew samples (from 0.01+/-0.00 to 0.29+/-0.01 mg/kg). Total phenol content (TP) and total protein (TProt) were comparable to those in literature. Multivariate analysis indicated that N-NO groups were significantly associated with NO(2)(-) and TP thus to suggest an in situ environmental nitrosation of specific nitrosable substrates (lysine, proline) favored by high reducing conditions. The bee-smoking process can be an alternative or complementary explanation for N-NO contamination. Hence NO(3)(-) rich honeys intake may exert beneficial effects against NSAIDs-induced gastric injury. Finally NO(3)(-) is a potential reliable marker of a honeys origin and quality.

Ionic profile of honey as a potential indicator of botanical origin and global environmental pollution

Aim of this study was to determine by Ion Chromatography ions (Na(+), Ca(++), Mg(++), NH4(+), Cl(-), Br(-), SO4(2-), NO3(-), PO4(3-)) in honeys (honeydew and floral nectar honeys) from different Italian Regions and from countries of the Western Balkan area. The compositional data were processed by multivariate analysis (PCA and HCA). Arboreal honeydew honeys from the Western Balkans had higher concentrations (from two to three times) of some environmental pollutants (Br(-), SO4(2-) and PO4(3-) contents), due to industrial and agricultural activities, than those from Italian regions. The cationic profiles were very similar in both groups. Multivariate analysis indicated a clear difference between nectar honeys and arboreal/honeydew honeys (recognition of the botanical origin). These findings point to the potential of ionic constituents of honey as indicators of environmental pollution, botanical origin and authenticity.

Preservation of endothelium nitric oxide release during beating heart surgery with respect to continuous flow cardiopulmonary bypass

A correlation between perfusion modality and vascular dilation induced by endothelial nitric oxide (NO) release has been pointed out in the literature; nevertheless, only a few studies deal with the analysis of patients treated by cardiac surgery. The aim of this work is to analyze endothelial NO release in patients undergoing cardiac surgery under continuous flow cardiopulmonary bypass (CPB) or pulsatile perfusion. Pulsatile devices approved for clinical CPB do not accurately reproduce the physiological flow waveform provided by the left ventricle while, on the other hand, it is important to analyze pulsatile perfusion under both physiological flow waveform and pulsatile flow CPB. Physiological pulsatile perfusion (supplied by the left ventricle) was examined in this study. A total of 16 patients undergoing cardiac surgery were enrolled in the study and divided into two groups: 8 patients were put on continuous flow CPB while the others underwent beating heart surgery. Venous blood samples were withdrawn to quantify endothelial NO release through its bioactive forms in blood. Plasma was used for the chemiluminescent detection of nitrite (NO2 —) and nitrate (NO3 —), and the cellular component for electron spin resonance detection of nitrosylhemoglobin. Significant reduction in the intraoperative concentration with respect to the preoperative was observed only in the continuous group for both NO2 — and NOx (NO2 — + NO3 —) concentration (p=0.003 and p=0.016, respectively). A significant difference in the intraoperative nitrite concentration was also observed between the groups (p=0.006). Nitrosylhemoglobin concentration, although not instrumentally detectable, resulted as negligible with respect to the other NO metabolites. Despite the small number of patients belonging to each group, this significant reduction of NO2 — concentration under continuous flow CPB revealed a strong dependence on endothelial NO release and plasma nitrite concentration on perfusion modality.

Unsaponifiable Fraction of Unripe Fruits of Olea europaea: An Interesting Source of Anti-inflammatory Constituents.

The unsaponifiable fraction of olive oil from unripe fruits of Olea europaea at different stages of maturation (from 20 to 32 weeks after flowering) was analyzed by gas chromatography-mass spectrometry in order to select the time associated to the unsaponifiable fraction with the maximal yield in bioactive constituents. According to quantitative gas chromatography-mass spectrometry analysis, the unsaponifiable fraction (2.46% of the total oil) from olive fruits at the 22nd week was found to contain the maximal yield in anti-inflammatory constituents. Its composition was lanosterol (2.60 mg/g oil), stigmasterol (2.15), cycloartanol acetate (2.04), stigmastan-3,5-diene (2.01), obtusifoliol (1.93), cholesta-4,6-dien-3-one (1.42), α-amyrin (1.42), α-tocopherol (1.32), squalene (1.02), β-amyrin (0.57), and β-sitosterol (0.22). At later times, there was a decrease in the quantitative unsaponifiable fraction yield and a qualitative shift in the bioactive constituents. The 22nd week unsaponifiable fraction was subsequently incorporated into a topical preparation to be utilized for a small pilot clinical study in five patients affected by osteoarthrosis. According to clinical observation, the application of the ointment (three times daily for three weeks) attenuated hand and knee joint inflammatory features in all patients and was not associated to any adverse reactions.

GC-MS characterisation and biological activity of essential oils from different vegetative organs of Plectranthus barbatus and Plectranthus caninus cultivated in north Italy

Essential oils (EOs) from the roots, stems and leaves of Plectranthus barbatus (A) and Plectranthuscaninus (B), cultivated in north Italy, were obtained by steam distillation and chemically characterised by gas chromatography–mass spectrometry. The highest yields were obtained from roots (268.15 and 673.60 mg/kg from A and B), followed by leaves (64.34 and 26.65 mg/kg) and stems (19.76 and 18.63 mg/kg). A total of 128 structures were identified in A and 121 in B. Fe++ chelating and antiradical activities (DPPH and ABTS) were evaluated: root and stem EOs showed the strongest activities, while EOs from leaves did not show relevant activities. All EOs were tested for their in vitro antimicrobial activity, showing optimal growth-inhibition in antibiogram (∅>35 mm) and MIC tests (32–64 μg/mL) against Candida albicans, while EOs from leaves of both species showed a good activity (25 < ∅ < 34 mm, MIC 64–128 μg/mL) against Escherichia coli.

A simplified screening procedure for determination of total N-NO groups (TNG) and nitrite (NO2-) in commercial low-molecular-weight heparins (LMWH) by selective chemical denitrosation followed by high-sensitivity chemiluminescence detection (NO-analyzer, NOA).

Aim of this work was to set up a method for the sensitive and selective determination of nitrite (NO(2)(-)) and total N-nitroso groups (TNG) in dalteparin and nadroparin, commercial low- molecular-weight heparins (LMWH), prepared by deaminative depolymerization of heparin with nitrous acid. The European Pharmacopoeia VI ed. indicates respectively 5 ppm as the maximum content for contaminant NO(2)(-) in the former and 0.25 ppm for TNG in the latter and no clear indication is given for N-NO groups in dalteparin, i.e. TNG must be absent because of the specific manufacturing process. The proposed technique is based on the development of a pre-analytical device, coupled to a chemiluminometer, constituted by three sequentially connected and commercially available purge vessels, where selective reagents are employed for the conversion of NO(2)(-) and N-NO to nitric oxide (NO). In detail, NO(2)(-) was determined in the first chamber and non-volatile and volatile TNG in the second and third. This method was validated for selectivity, sensitivity, linearity, accuracy and precision. The method was shown to be selective, with a quantitative linear range of 1-1000 ppb). The bias, intra- and inter-day percent relative error was lower than 1%. The contamination of NO(2)(-) and TNG in nadreparin was below the limits; for dalteparin NO(2)(-) fell within the limit, but there was a huge amount of TNG (15.80+/-0.05 ppm-6.69+/-0.02 ppm). Preliminary investigation on the solvent-extractable material from dalteparin showed the majority of chemiluminescence retained in the aqueous residue to indicate that this N-NO groups may belong to solvent unextractable material or be tightly bound to the dalteparin backbone.

Air dispersed essential oils combined with standard sanitization procedures for environmental microbiota control in nosocomial hospitalization rooms

OBJECTIVE Environmental bacterial contaminant microorganisms are an ongoing problem in hospitals. Essential oil vapours (EO) may help reducing this type of contamination. Aim of this study was to evaluate the efficacy of nebulized selected essential oils (EO) in reducing the microbial contamination in residential health care house rooms. DESIGN The study was carried out in a two-story 112-bed tertiary care structure (approximately 1060 m(2)). Contamination in rooms and corridors was monitored for a total of n=5 months, including a starting baseline sampling and one end-study point, and without combined treatment (standard sanitization alone). Contact slides were collected for microbiological analysis. RESULTS Reductions in both bacterial and fungal contamination were observed between rooms cleaned using standard sanitization alone or in combination with essential oils nebulization (average 90% decrease for total count, P<0.01; 90% for yeasts and molds, P<0.05). Decreases of antibiotic (70%), mucolytic (100%), bronchodilators (100%), and steroidal (67%) and non-steroidal anti-inflammatory drugs (33%) prescriptions were observed, with no adverse effects on patients. CONCLUSIONS The selected EO composition is effective in reducing both the environmental microbial contamination and pharmaceutical drugs consumption in a nosocomial health care house. This study demonstrates that aerial EO diffusion combined with standard sanitization procedures, has great potential to reduce the microbial contamination in critical hospital environments such as hospitalization rooms.