Is the Antimicrobial Activity of Hydrolates Lower than That of Essential Oils?

Among the top five human infections requiring medical treatment is dermatitis. Treatment of bacterial and fungal skin infections is usually based on antibiotic therapy, which is often ineffective due to the involvement of antibiotic-resistant microbial strains. The aim of this study was to compare the antimicrobial activity of essential oils (EOs) and hydrolates (Hys) extracted from six aromatic plants grown in Italy (Lavandula angustifolia, Lavandula intermedia, Origanum hirtum, Satureja montana, Monarda didyma, and Monarda fistulosa) towards fungal (Candida albicans, Candida parapsilosis, Candida glabrata and Candida tropicalis; Trichophyton soudanense, Trichophyton tonsurans, Trichophyton rubrum, Trichophyton violaceum and Microsporum canis) and bacterial strains (Staphylococcus aureus MRSA, Staphylococcus aureus MSSA, Streptococcus pyogenes, E. faecalis, Enterococcus faecalis VRE, and Enterococcus faecium) potentially pathogenic for human skin. The composition and antimicrobial activity of EOs and Hys were evaluated using the Gas-chromatography mass spectrometry and micro dilution-broth test, respectively. The volatiles' conversion factors (CFs) were calculated to compare the activity of Hys with that of the corresponding EOs. Data show that, although the minimum inhibitory concentration values of EOs are lower than the corresponding Hys, the volatiles contained in Hys are more effective at inhibiting microbial growth because they are active at lower concentrations.

Beet on Alps: Time-course changes of plasma nitrate and nitrite concentrations during acclimatization to high-altitude.

Nitric oxide seems to be involved in the altitude acclimatization process due to its ability to regulate pulmonary, cardiovascular and muscular responses to hypoxia. In this study, we investigated the plasma nitrate (NO3-) and nitrite (NO2-) response to hypobaric hypoxia in two groups of lowlanders exposed at different altitudes. For seven days, fourteen subjects were evaluated at Casati Hut (3269 m a.s.l. M.CEVEDALE) and eleven individuals were studied at Capanna Regina Margherita (4554 m a.s.l. M.ROSA). Before expeditions and at different time points during high-altitude sojourn, plasma NO3- and NO2- concentrations were measured by chemiluminescence. Resting peripheral arterial oxygen saturation (SpO2), heart rate (HR) and mean arterial blood pressure (MAP) were monitored during the experimental period. Possible confounding factors such as dietary NO3- intake, physical activity and altitude changes were controlled. Sea level plasma NO3- and NO2- concentrations significantly increased at altitude in both M.CEVEDALE group (+26.2 µM, p ≤ 0.0001, 95% CI [+17.6, +34.8] and +559.2 nM, p ≤ 0.0001, [+332.8, +785.6]) and M.ROSA group (+18.7 µM, p ≤ 0.0001, [+10.8, +26.5] and +463.7 nM, p ≤ 0.0001, [+314.3, +613.0]). Average peak value in NO metabolites concentration occurred earlier in M.CEVEDALE group vs M.ROSA group (NO3-, day 3 vs day 5, p = 0.007; NO2-, day 3 vs day 5, p = 0.019). In both groups, resting SpO2, HR and MAP values changed according to altitude levels. This study shows that exposure to hypobaric hypoxia affects nitric oxide metabolites, resulting in a significant increase in plasma NO3- and NO2- concentrations from sea level values. Interestingly, the higher the altitude reached, the longer the time taken to reach a peak in plasma concentrations of nitric oxide metabolites.

GC-MS analysis and biological activity of hydroalcholic extracts and essential oils of Rhus typhina L. wood (anacardiaceae) in comparison with leaves and fruits.

Hydroalcoholic extracts (HE) and essential oils (EO) of branches, leaves and fruits of Rhus typhina L., were characterized by GC-MS. HE (yealds: branches 68.30 mg/g, leaves 35.82 mg/g and fruits 257.76 mg/g), showed different compositions dominated by gallic acid (33.46%) in branches, its precursor 1-cyclohexane-3,4,5-hydroxy-carboxylic acid (20.55%) in leaves and malic acid (89.15%) in fruits. EO yields were 210 µg/g for branches (main component δ-cadinene, 22.00%), followed by fruits with 132 µg/g (ß-pinene 32.2%) and by leaves with 54 µg/g and phenylacetaldehyde as major component (40.13%). Total phenolic content (TPC) was highest in branches HE (5.87 µg GAE/mL) and in EO leaves (17.71 µg GAE/mL). The highest value of radical scavenging activity (DPPH test) was detected in leaves HE and EO. The branches EO antimicrobial activity was strong against C. albicans and negligible against E. coli. Leaves and fruits EO showed strong activity against C. albicans and intermediate activity against Escherichia coli.

Oxidative Stress Modulation by Cameroonian Spice Extracts in HepG2 Cells: Involvement of Nrf2 and Improvement of Glucose Uptake.

Oxidative stress plays a relevant role in the progression of chronic conditions, including cardiometabolic diseases. Several Cameroonian plants, including spices, are traditionally used as herbal medicines for the treatment of diseases where oxidative stress contributes to insulin resistance, like type 2 diabetes mellitus. This study evaluated the antioxidant capacity and the effects on oxidative-stress-induced impairment of glucose uptake of 11 Cameroonian spice extracts. H2O2-induced reactive oxygen species (ROS) production by human HepG2 cells was significantly reduced by 8/11 extracts. The most effective extracts, Xylopia parviflora, Echinops giganteus, and Dichrostachys glomerata, showed a concentration-dependent ROS-scavenging activity, which involved Nrf2 translocation into the nucleus. Xylopia parviflora, Tetrapleura tetraptera, Dichrostachys glomerata, Aframomum melegueta, and Aframomum citratum extracts showed the highest antioxidant capacity, according to oxygen radical absorbance capacity (ORAC) (2.52-88 µM Trolox Eq/g of extract), ferric-reducing antioxidant power (FRAP) (40.23-233.84 mg gallic acid Eq/g of extract), and total phenol (8.96-32.96% mg gallic acid Eq/g of extract) assays. In HepG2 cells, glucose uptake was stimulated by 4/11 extracts, similarly to insulin and metformin. H2O2-induced oxidative stress reduced glucose uptake, which was rescued by pretreatment with Xylopia aethiopica, Xylopia parviflora, Scorodophloeus zenkeri, Monodora myristica, and Dichrostachys glomerata extracts. The ROS-scavenging ability of the spice extracts may reside in some secondary metabolites observed by phytochemical profiling (reverse-phase high-performance liquid chromatography coupled to a diode array detector (HPLC-UV-DAD)). Further studies are needed to better clarify their biological activities and potential use to control oxidative stress and promote insulin sensitivity.

Evaluation of antimicrobial activity of the hydrolate of Coridothymus capitatus (L.) Reichenb. fil. (Lamiaceae) alone and in combination with antimicrobial agents.

BACKGROUND: Hydrolates, complex mixtures containing traces of essential oils (EOs), are inexpensive, easy to make and less toxic than their corresponding EOs. The antibacterial and antifungal activity of the hydrolate of Coridothymus capitatus (L.) Reichenb. fil. (Lamiaceae) alone and in combination with antimicrobial drugs, such as tetracycline and itraconazole, were evaluated. METHODS: The chemical composition was analysed by gas-chromatography-mass spectrometry (GC-MS). Standard methods were performed to evaluate the susceptibility of some Gram-positive and Gram-negative bacteria, and Candida spp. to the hydrolate, in comparison with its EO. The hydrolate mechanism of action was assayed by propidium iodide and MitoTracker staining. Checkerboard tests were carried out for combinations studies. RESULTS: GC-MS identified 0.14% (v/v) of total EO content into hydrolate and carvacrol as a dominant component. The hydrolate showed a good antimicrobial activity against bacteria and yeasts. It exhibited a synergistic effect with itraconazole against Candida krusei, and an additive effect with tetracycline against methicillin-resistant Staphylococcus aureus strains. Hydrolate changed the membranes permeability of bacteria and yeasts and altered mitochondrial function of yeasts. CONCLUSIONS: Our study extends the knowledge by exploiting non-conventional antimicrobial agents to fight the emergence of antibiotic resistance.

Hydroethanolic plant extracts from Cameroon positively modulate enzymes relevant to carbohydrate/lipid digestion and cardio-metabolic diseases.

Cardiovascular diseases are the greatest cause of death globally and are frequently associated with type 2 diabetes mellitus and metabolic syndrome, a condition including visceral obesity, hypertension, elevated triglycerides and low HDL cholesterol and hyperglycaemia. Several medicinal plants, including spices, are used in Cameroon as herbal medicines and are traditionally employed for the treatment of several ailments such as diabetes and related diseases. In this study, we chemically characterized eleven Cameroonian spice extracts and evaluated their effects on some enzyme activities relevant to carbohydrate and lipid digestion and cardio-metabolic diseases. Hydroethanolic spice extracts were characterized by GC-MS analysis and screened for their ability to modulate the activity of α-glucosidase, α-amylase, pancreatic lipase, 3-hydroxy-3-methylglutaryl-coenzyme A reductase and angiotensin-converting enzyme (ACE). Among the spice extracts tested, those from Xylopia parviflora showed the widest inhibitory spectrum, with a relevant effect on all enzyme activities. Dichrostachys glomerata and Aframomum citratum extracts were more selective. The selected and strong activity of some plants, such as that of Aframomum citratum on pancreatic lipase and that of Xylopia aethiopica on ACE, suggests their specific use in obesity and hypertension, respectively. Chemical analysis indicated that for some spice extracts such as Xylopia parviflora and Aframomum citratum their secondary metabolites (chlorogenic acid, pimaric acid, and catechin and its derivatives) could potentially justify the biological properties observed. Our findings clearly show significant inhibition of cardio-metabolic enzymes by hydroethanolic Cameroonian spice extracts, suggesting the potential usefulness of nutraceuticals derived from these plants to develop novel management strategies for obesity and diabetes complications.

Identification by Molecular Docking ofHomoisoflavones from Leopoldia comosa as Ligands of Estrogen Receptors.

The physiological responses to estrogen hormones are mediated within specific tissues by at least two distinct receptors, ER and ER. Several natural and synthetic molecules show activity by interacting with these proteins. In particular, a number of vegetal compounds known as phytoestrogens shows estrogenic or anti-estrogenic activity. The majority of these compounds belongs to the isoflavones family and the most representative one, genistein, shows anti-proliferative effects on various hormone-sensitive cancer cells, including breast, ovarian and prostate cancer. In this work we describe the identification of structurally related homoisoflavones isolated from Leopoldia comosa (L.) Parl. (L. comosa), a perennial bulbous plant, potentially useful as hormonal substitutes or complements in cancer treatments. Two of these compounds have been selected as potential ligands of estrogen receptors (ERs) and the interaction with both isoforms of estrogen receptors have been investigated through molecular docking on their crystallographic structures. The results provide evidence of the binding of these compounds to the target receptors and their interactions with key residues of the active sites of the two proteins, and thus they could represent suitable leads for the development of novel tools for the dissection of ER signaling and the development of new pharmacological treatments in hormone-sensitive cancers.

Semi-preparative HPLC purification of δ-tocotrienol (δ-T3) from Elaeis guineensis Jacq. and Bixa orellana L. and evaluation of its in vitro anticancer activity in human A375 melanoma cells.

In this work, we report a rapid and convenient HPLC-UV-DAD method for the isolation of δ-T3 on semi-preparative scale from two different vitamin E rich processed, commercially available products obtained from the fruits of Elaeis guineensis Jacq. (oil palm) and from the seeds of Bixa orellana L. (achiote tree). Chromatography was run using reverse phase (RP) C-18 columns and HPLC-grade acetonitrile as mobile phase. The purity of the isolated δ-T3, assessed by GC-MS and 1H NMR was above 98%. The δ-T3 cytotoxic activity found in vitro against the proliferation of human A375 melanoma cells compared to that of the other δ-T3 free tocols mixture suggest its primary role in the experimental anticancer activity observed for palm oil derived products. Taken altogether, the results of this study highlight the importance of the application of suitable purification systems for the preparations of tocotrienols prior to their experimental or clinical testing.

Comprehensive quality evaluation of medical Cannabis sativa L. inflorescence and macerated oils based on HS-SPME coupled to GC-MS and LC-HRMS (q-exactive orbitrap®) approach.

There are at least 554 identified compounds in C. sativa L., among them 113 phytocannabinoids and 120 terpenes. Phytocomplex composition differences between the pharmaceutical properties of different medical cannabis chemotype have been attributed to strict interactions, defined as 'entourage effect', between cannabinoids and terpenes as a result of synergic action. The chemical complexity of its bioactive constituents highlight the need for standardised and well-defined analytical approaches able to characterise the plant chemotype, the herbal drug quality as well as to monitor the quality of pharmaceutical cannabis extracts and preparations. Hence, in the first part of this study an analytical procedures involving the combination of headspace-solid-phase microextraction (HS-SPME) coupled to GC-MS and High Resolution Mass-Spectrometry LC-HRMS (Orbitrap®) were set up, validated and applied for the in-depth profiling and fingerprinting of cannabinoids and terpenes in two authorised medical grade varieties of Cannabis sativa L. inflorescences (Bedrocan® and Bediol®) and in obtained macerated oils. To better understand the trend of all volatile compounds and cannabinoids during oil storage a new procedure for cannabis macerated oil preparation without any thermal step was tested and compared with the existing conventional methods to assess the potentially detrimental effect of heating on overall product quality.

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.

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), ß-amyrin (0.57), and ß-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.

A novel truncated form of eNOS associates with altered vascular function.

AIMS: Nitric oxide (NO) plays a key role in vascular homeostasis and is produced by endothelial NO synthase (eNOS), encoded by NOS3 gene. We previously reported the genetic association between NOS3 rs753482-A>C polymorphism on intron 19 and coronary artery disease (CAD). In the attempt of conferring functional implication to the rs753482-A>C polymorphism, we investigated its influence on transcript maturation. METHODS AND RESULTS: A transcript variant skipping exons 20-21 is prevalent in carriers of the rs753482-C allele and is translated in a novel truncated form of eNOS. The truncated eNOS displays increased basal NO production, is insensitive to calcium stimulation, and, upon heterodimerization with the full-length eNOS protein, exerts a dominant-negative effect on NO production. CAD patients and healthy subjects' carriers of the rs753482-C genotype are characterized by increased NO basal levels in peripheral blood and platelets, and negatively respond to oral glucose load by failing to increase NO synthesis following insulin wave. Furthermore, forearm vasodilation after reactive hyperaemia is dramatically impaired in rs753482-C carriers. CONCLUSIONS: We demonstrated that subjects carrying the rs753482-C genotype express a novel stable truncated form of eNOS with altered enzymatic activity that influences NO production and endothelial function. These findings open to new intriguing perspectives to several diseases involving vascular response to NO.

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.

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%), ß-selinene (5.00%) and ß-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-1ß, 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.

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 honey's origin and quality.

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 (NO(2)(-)) and nitrate (NO(3)(-)), 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 NO(2)(-) and NO(x) (NO(2)(-) + NO(3)(-)) 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 NO(2)(-) concentration under continuous flow CPB revealed a strong dependence on endothelial NO release and plasma nitrite concentration on perfusion modality.

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.

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.