Hypoglycemic active principles from the leaves of Bauhinia holophylla: Comprehensive phytochemical characterization and in vivo activity profile.

Bauhinia holophylla leaves, also known as "pata-de-vaca", are traditionally used in Brazil to treat diabetes. Although the hypoglycemic activity of this medicinal plant has already been described, the active compounds responsible for the hypoglycemic activity have not yet been identified. To rapidly obtain two fractions in large amounts compatible with further in vivo assay, the hydroalcoholic extract of B. holophylla leaves was fractionated by Vacuum Liquid Chromatography and then purified by medium pressure liquid chromatography combined with an in vivo Glucose Tolerance Test in diabetic mice. This approach resulted in the identification of eleven compounds (1-11), including an original non-cyanogenic cyanoglucoside derivative. The structures of the isolated compounds were elucidated by nuclear magnetic resonance and high-resolution mass spectrometry. One of the major compounds of the leaves, lithospermoside (3), exhibited strong hypoglycemic activity in diabetic mice at the doses of 10 and 20 mg/kg b.w. and prevents body weight loss. The proton nuclear magnetic resonance (1H NMR) quantification revealed that the hydroalcoholic leaves extract contained 1.7% of lithospermoside (3) and 3.1% of flavonoids. The NMR analysis also revealed the presence of a high amount of pinitol (4) (9.5%), a known compound possessing in vivo hypoglycemic activity. The hypoglycemic properties of the hydroalcoholic leaves extract and the traditional water infusion extracts of the leaves of B. holophylla seem thus to be the result of the activity of three unrelated classes of compounds. Such results support to some extent the traditional use of Bauhinia holophylla to treat diabetes.

Niazirin from Moringa oleifera Lam. attenuates high glucose-induced oxidative stress through PKCζ/Nox4 pathway.

BACKGROUND: Diabetic complications-coronary atherosclerosis is closely related to the increased reactive oxygen species (ROS) induced by hyperglycemia. ROS are reported to induce the abnormal proliferation of vascular smooth muscle cells (VSMCs) under high glucose conditions. Leaf and seed extracts from Moringa oleifera are found to exhibit antioxidant activity. However, few studies are evaluating the antioxidant activities of chemical compounds isolated from the M. oleifera especially in cardiovascular field. PURPOSE: The aim of this study is to explore the antioxidative effect during hyperglycemia of niazirin from M. oleifera. STUDY DESIGN: A cell model was applied. METHODS: After the taking the in vitro antioxidant experiment including ferric reducing antioxidant power (FRAP), 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) assay and 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. Cell viability was carried out using high glucose-induced VSMCs model. ROS production was tested by 2',7'-dichlorofluorescein diacetate (DCF-DA) assay. The protein kinase C zeta (PKCζ) and NADPH oxidase 4 (Nox 4) expression in vitro and in vivo were measured by western blot analysis. RESULTS: Niazirin showed good free radical scavenging activity. Niazirin significantly attenuated the proliferation of high glucose-induced VSMCs. Furthermore, it could decrease the ROS and malondialdehyde (MDA) productions, while increased total antioxidant capacity (T-AOC), superoxide dismutase (SOD) as well as glutathione peroxidase (GPx) levels in high glucose-induced VSMCs and streptozotocin-induced mice. In addition, niazirin could eliminate the high glucose-induced PKCζ activation, indicated by Thr410 phosphorylation and inhibition of the Nox4 protein expression in vitro and in vivo. CONCLUSION: Niazirin from M. oleifera exhibited notably antioxidant activities and could be utilized as a potential natural antioxidant in preventing diabetic atherosclerosis.

Innate positive chemotaxis to paeonal from highly attractive Chinese medicinal herbs in the cigarette beetle, Lasioderma serricorne.

Lasioderma serricorne, also known as cigarette beetle, can exploit a wide variety of stored materials as foods, but it is particularly common on tobacco and herbs. This beetle is a dominant pest species of stored Chinese medicinal materials (CMMs) causing high economic damages, making effective control strategies urgently needed. Behavioural manipulation is an important component of Integrated Pest Management. To the best of our knowledge, plant-borne volatile organic compounds (VOCs) have never been explored to develop lures for managing L. serricorne. In this study, the behavioural responses of L. serricorne to VOCs from four selected CMMs (Euphorbia kansui, Aconitum carmichaelii, Eucommia ulmoides and Pinellia ternata) were studied and their components analysed. Then, the olfactory responses of L. serricorne to the most abundant VOC identified in the preferred CMM, i.e., paeonal, was tested. L. serricorne showed significant differences in its preferences for the VOCs from the four CMMs, i.e, E. kansui > A. carmichaelii > E. ulmoides > P. ternata. From the VOCs of E. kansui, A. carmichaelii, E. ulmoides, and P. ternata, 77, 74, 56, and 81 molecules, were identified, respectively. Paeonal (23.5%), junipene (17.2%), hexanal (17.1%), and benzeneacetonitrile (14.0%) were the most abundant, respectively. Since paeonal dominated the VOC spectrum of the most preferred CMM, this compound was selected for further studies. L. serricorne showed significant positive responses to paeonal tested at various doses, with the most attractive ones being 100 µg and 500 µg. Our findings shed light on the olfactory cues routing the food searching behaviour in the cigarette beetle, providing important information on how L. serricorne targets particular CMMs. The high attractiveness of paeonal at low doses tested here may be exploited further to develop novel monitoring and control tools (e.g., lure-and-kill strategies) against this important stored product pest.

Comparative Drug Resistance Reversal Potential of Natural Glycosides: Potential of Synergy Niaziridin & Niazirin.

BACKGROUND: Due to the limited availability of antibiotics, Gram-negative bacteria (GNB) acquire different levels of drug resistance. It raised an urgent need to identify such agents, which can reverse the phenomenon of drug resistance. OBJECTIVE: To understand the mechanism of drug resistance reversal of glycosides; niaziridin and niazirin isolated from the pods of Moringa oleifera and ouabain (control) against the clinical isolates of multidrug-resistant Escherichia coli. METHODS: The MICs were determined following the CLSI guidelines for broth micro-dilution. In-vitro combination studies were performed by broth checkerboard method followed by Time-Kill studies, the efflux pump inhibition assay, ATPase inhibitory activity, mutation prevention concentration and in-silico studies. RESULTS: The results showed that both glycosides did not possess antibacterial activity of their own, but in combination, they reduced the MIC of tetracycline up to 16 folds. Both were found to inhibit efflux pumps, but niaziridin was the best. In real time expression pattern analysis, niaziridin was also found responsible for the down expression of the two important efflux pump acrB & yojI genes alone as well as in combination. Niaziridin was also able to over express the porin forming genes (ompA & ompX). These glycosides decreased the mutation prevention concentration of tetracycline. CONCLUSION: This is the first ever report on glycosides, niazirin and niaziridin acting as drug resistance reversal agent through efflux pump inhibition and modulation of expression pattern drug resistant genes. This study may be helpful in preparing an effective antibacterial combination against the drug-resistant GNB from a widely growing Moringa oleifera.

Anti-Tumorigenic and Anti-Metastatic Activity of the Sponge-Derived Marine Drugs Aeroplysinin-1 and Isofistularin-3 against Pheochromocytoma In Vitro.

Over 10% of pheochromocytoma and paraganglioma (PPGL) patients have malignant disease at their first presentation in the clinic. Development of malignancy and the underlying molecular pathways in PPGLs are poorly understood and efficient treatment strategies are missing. Marine sponges provide a natural source of promising anti-tumorigenic and anti-metastatic agents. We evaluate the anti-tumorigenic and anti-metastatic potential of Aeroplysinin-1 and Isofistularin-3, two secondary metabolites isolated from the marine sponge Aplysina aerophoba, on pheochromocytoma cells. Aeroplysinin-1 diminished the number of proliferating cells and reduced spheroid growth significantly. Beside these anti-tumorigenic activity, Aeroplysinin-1 decreased the migration ability of the cells significantly (p = 0.01), whereas, the invasion capacity was not affected. Aeroplysinin-1 diminished the high adhesion capacity of the MTT cells to collagen (p < 0.001) and, furthermore, reduced the ability to form spheroids significantly. Western Blot and qRT-PCR analysis showed a downregulation of integrin ß1 that might explain the lower adhesion and migration capacity after Aeroplysinin-1 treatment. Isofistularin-3 showed only a negligible influence on proliferative and pro-metastatic cell properties. These in vitro investigations show promise for the application of the sponge-derived marine drug, Aeroplysinin-1 as anti-tumorigenic and anti-metastatic agent against PPGLs for the first time.

Beneficial Effect of Jojoba Seed Extracts on Hyperglycemia-Induced Oxidative Stress in RINm5f Beta Cells.

Hyperglycemia occurs during diabetes and insulin resistance. It causes oxidative stress by increasing reactive oxygen species (ROS) levels, leading to cellular damage. Polyphenols play a central role in defense against oxidative stress. In our study, we investigated the antioxidant properties of simmondsin, a pure molecule present in jojoba seeds, and of the aqueous extract of jojoba seeds on fructose-induced oxidative stress in RINm5f beta cells. The exposure of RINm5f beta cells to fructose triggered the loss of cell viability (-48%, p < 0.001) and disruption of insulin secretion (p < 0.001) associated with of reactive oxygen species (ROS) production and a modulation of pro-oxidant and antioxidant signaling pathway. Cell pre-treatments with extracts considerably increased cell viability (+86% p < 0.001) for simmondsin and +74% (p < 0.001) for aqueous extract and insulin secretion. The extracts also markedly decreased ROS (-69% (p < 0.001) for simmondsin and -59% (p < 0.001) for aqueous extract) and caspase-3 activation and improved antioxidant defense, inhibiting p22phox and increasing nuclear factor (erythroid-derived 2)-like 2 (Nrf2) levels (+70%, p < 0.001) for aqueous extract. Simmondsin had no impact on Nrf2 levels. The richness and diversity of molecules present in jojoba seed extract makes jojoba a powerful agent to prevent the destruction of RINm5f beta cells induced by hyperglycemia.

Effective purification of recombinant peptide ligands for GPCR research.

Peptide purification from natural sources and chemical synthesis is cumbersome with various shortcomings such as low yield, high cost of production, error prone, and restricted by nature of amino acids. Though recombinant DNA technology had overcome all these setbacks for larger proteins, it is still a challenge to produce peptides that are salt free and without impurities. Our approach discussed in this chapter deals with easy and effective purification of peptides of varying sizes (up to 10kDa), expressed as fusion proteins in bacterial system. This includes cleavage of fusion affinity tag by "PreScission protease" in volatile buffer followed by selective acetonitrile precipitation of high-molecular-weight tag in order to purify peptides in solution. This method can be used to purify peptides in large scale for various biochemical and physiological studies.

The shadow of dichloroacetonitrile (DCAN), a typical nitrogenous disinfection by-product (N-DBP), in the waterworks and its backwash water reuse.

Dichloroacetonitrile (DCAN) is one of nitrogenous disinfection by-products (N-DBPs) with strong cytotoxicity and genotoxicity. In this study, the formation potential (FP) of DCAN was investigated in the samples of six important water sources located in the Yangtze River Delta. The highest formation concentration of DCAN was 9.05 µg/L in the water sample taken from Taihu Lake with the lowest SUVA value. After the NOM fractionation, the conversion rate of hydrophilic fraction to DCAN was found the highest. Subsequently, a waterworks using Taihu Lake as water source was chosen to research the FP variations of DCAN in the treatment process and backwash water. The results showed that, compared to the conventional treatment process, O/biological activated carbon (BAC) process increased the removal efficiency of DCAN from 21.89% to 50.58% by removing aromatic protein and soluble biological by-products as main precursors of DCAN. The DCAN FP in the effluent of BAC filters using old granular activated carbon was higher than that in the influent and the DCAN FP of its backwash water was lower than that in raw water. In the backwash water of sand filters, the DCAN FP higher than raw water required the recycle ratio less than 5% to avoid the accumulation of DCAN.

Formation and speciation of haloacetamides and haloacetonitriles for chlorination, chloramination, and chlorination followed by chloramination.

The formation of haloacetamides (HAcAms) and haloacetonitriles (HANs) from a solution containing natural organic matter and a secondary effluent sample was evaluated for disinfection by chlorination, chloramination, and chlorination followed by chloramination (Cl2NH2Cl process). The use of preformed monochloramine (NH2Cl) produced higher concentrations of HAcAms and lower concentrations of HANs than chlorination, while the Cl2NH2Cl process produced the highest concentrations of HAcAms and HANs. These results indicate that the Cl2NH2Cl process, which inhibited the formation of regulated trihalomethanes compared with chlorination, enhanced the formation of HAcAms and HANs. For disinfection in the presence of bromide, brominated dihaloacetamides and dihaloacetonitriles were formed, and the trends were similar to those observed for chlorinated species in the absence of bromide. The degrees of bromine substitution of dihaloacetamides and dihaloacetonitriles were highest for chlorination, followed by the Cl2NH2Cl process and then by the NH2Cl process. For the Cl2NH2Cl process, HAN formation kept gradually increasing with prechlorination time increasing from 0 to 120 min, while HAcAm formation increased only until it reached a maximum at around 10-30 min. These results suggest that the prechlorination time could be reduced to control the formation of HAcAms and HANs. During chloramination, the formation of HAcAms and HANs was lower when using preformed NH2Cl than when chloramines were formed in situ, with higher formation of HAcAms and HANs when chlorine was added before ammonia than vice versa for the secondary effluent; this finding suggests that preformed NH2Cl could be used to inhibit the formation of HAcAms and HANs during chloramination.

Acetonitrile extraction and dual-layer solid phase extraction clean-up for pesticide residue analysis in propolis.

Propolis is a very complex mixture of substances that is produced by honey bees and is known to be a rather challenging matrix for residue analysis. Besides resins, flavonoids and phenols, high amount of wax is co-extracted resulting in immense matrix effects. Therefore a suitable clean-up is crucial and indispensable. In this study, a reliable solid phase extraction (SPE) clean-up was developed for pesticide residue analysis in propolis. The clean-up success was quickly and easily monitored by high-performance thin-layer chromatography with different detection possibilities. The final method consists of the extraction of propolis with acetonitrile according to the QuEChERS method followed by an effective extract purification on dual-layer SPE cartridges with spherical hydrophobic polystyrene-divinylbenzene resin/primary secondary amine as sorbent and a mixture of toluene/acetone (95:5, v/v) for elution. Besides fat-soluble components like waxes, flavonoids, and terpenoids, more polar compounds like organic acids, fatty acids, sugars and anthocyanins were also removed to large extent. Method performance was assessed by recovery experiments at spiking levels of 0.5 and 1mg/kg (n=5) for fourteen pesticides that are relevant for propolis. Mean recoveries determined by HPLC-MS against solvent standards were between 40 and 101%, while calculation against matrix-matched standards provided recoveries of 79-104%. Precision of recovery, assessed by relative standard deviations, were below 9%. Thus, the developed dual-layer SPE clean-up enables the reliable pesticide residue analysis in propolis and provides a suitable alternative to time-consuming clean-up procedures proposed in literature.

Four New Cyclohexylideneacetonitrile Derivatives from the Hypocotyl of Mangrove (Bruguiera gymnorrhiza).

Four new cyclohexylideneacetonitrile derivatives 1-4, named menisdaurins B-E, as well as three known cyclohexylideneacetonitrile derivatives--menisdaurin (5), coclauril (6), and menisdaurilide (7)--were isolated from the hypocotyl of a mangrove (Bruguiera gymnorrhiza). The structures of the isolates were elucidated on the basis of extensive spectroscopic analysis. Compounds 1-7 showed anti-Hepatitis B virus (HBV) activities, with EC50 values ranging from 5.1 ± 0.2 µg/mL to 87.7 ± 5.8 µg/mL.

Influence of activated carbon porosity and surface oxygen functionalities' presence on adsorption of acetonitrile as a simple polar volatile organic compound.

Based on series of porous carbon models, systematic Monte Carlo studies on the adsorption of acetonitrile (as a simple representative of polar volatile organic compounds) were performed. The influence of porosity and chemical composition of the carbon surface on CH3CN adsorption was studied and it was shown that both the factors influenced the adsorption mechanism. A decrease in the pore size and the introduction of oxygen surface groups led to a rise in adsorption energy and to an increase in the filling of accessible volume in the low-pressure part of the isotherm. However, from a practical point of view, it is easier to increase the adsorption by introducing polar groups on the carbon surface than by modifying the porosity.

Tenualexin, other phytoalexins and indole glucosinolates from wild cruciferous species.

In general, the chemodiversity of phytoalexins, elicited metabolites involved in plant defense mechanisms against microbial pathogens, correlates with the biodiversity of their sources. In this work, the phytoalexins produced by four wild cruciferous species (Brassica tournefortii, Crambe abyssinica (crambe), Diplotaxis tenuifolia (sand rocket), and Diplotaxis tenuisiliqua (wall rocket)) were identified and quantified by HPLC with photodioarray and electrospray mass detectors. In addition, the production of indole glucosinolates, biosynthetic precursors of cruciferous phytoalexins, was evaluated. Tenualexin, (=2-(1,4-dimethoxy-1H-indol-3-yl)acetonitrile), the first cruciferous phytoalexin containing two MeO substituents in the indole ring, was isolated from D. tenuisiliqua, synthesized, and evaluated for antifungal activity. The phytoalexins cyclobrassinin and spirobrassinin were detected in B. tournefortii and C. abyssinica, whereas rutalexin and 4-methoxybrassinin were only found in B. tournefortii. D. tenuifolia, and D. tenuisiliqua produced 2-(1H-indol-3-yl)acetonitriles as phytoalexins. Because tenualexin appears to be one of the broad-range antifungals occurring in crucifers, it is suggested that D. tenuisiliqua may have disease resistance traits important to be incorporated in commercial breeding programs.

Multiresidue pesticide analysis of botanical dietary supplements using salt-out acetonitrile extraction, solid-phase extraction cleanup column, and gas chromatography-triple quadrupole mass spectrometry.

Dietary supplements form an increasing part of the American diet, yet broadly applicable multiresidue pesticide methods have not been evaluated for many of these supplements. A method for the analysis of 310 pesticides, isomers, and pesticide metabolites in dried botanical dietary supplements has been developed and validated. Sample preparation involved acetonitrile:water added to the botanical along with anhydrous magnesium sulfate and sodium chloride for extraction, followed by cleanup with solid-phase extraction using a tandem cartridge consisting of graphitized carbon black (GCB) and primary-secondary amine sorbent (PSA). Pesticides were measured by gas chromatography-tandem mass spectrometry. Accuracy and precision were evaluated through fortifications of 24 botanicals at 10, 25, 100, and 500 µg/kg. Mean pesticide recoveries and relative standard deviations (RSDs) for all botanicals were 97%, 91%, 90%, and 90% and 15%, 10%, 8%, and 6% at 10, 25, 100, and 500 µg/kg, respectively. The method was applied to 21 incurred botanicals. Quinoxyfen was measured in hops (100-620 µg/kg). Tetraconazole (48 µg/kg), tetramethrin (15 µg/kg), methamidophos (50 µg/kg), and chlorpyrifos (93 µg/kg) were measured in licorice, mallow, tea, and tribulus, respectively. Quintozene, its metabolites and contaminants (pentachloroaniline, pentachlorobenzene, pentachloroanisole, and pentachlorothioanisole and hexachlorobenzene and tecnazene, respectively), with hexachlorocyclohexanes and DDT were identified in ginseng sources along with azoxystrobin, diazinon, and dimethomorph between 0.7 and 2800 µg/kg. Validation with these botanicals demonstrated the extent of this method's applicability for screening 310 pesticides in a wide array of botanical dietary supplements.

Immobilization of Rhodococcus rhodochrous BX2 (an acetonitrile-degrading bacterium) with biofilm-forming bacteria for wastewater treatment.

In this study, a unique biofilm consisting of three bacterial strains with high biofilm-forming capability (Bacillus subtilis E2, E3, and N4) and an acetonitrile-degrading bacterium (Rhodococcus rhodochrous BX2) was established for acetonitrile-containing wastewater treatment. The results indicated that this biofilm exhibited strong resistance to acetonitrile loading shock and displayed a typical spatial and structural heterogeneity and completely depleted the initial concentration of acetonitrile (800mgL(-1)) within 24h in a moving-bed-biofilm reactor (MBBR) after operation for 30days. The immobilization of BX2 cells in the biofilm was confirmed by PCR-DGGE. It has been demonstrated that biofilm-forming bacteria can promote the immobilization of contaminant-degrading bacteria in the biofilms and can subsequently improve the degradation of contaminants in wastewater. This approach offers a novel strategy for enhancing biological oxidation of toxic pollutants in wastewater.

Right-first-time isocratic preparative liquid chromatography-mass spectrometry purification.

Preparative RP-LCMS is widely used to purify compounds from crude reaction mixtures in drug discovery today. Method development for preparative Liquid Chromatography-Mass Spectrometry (LCMS) consists of finding the right balance between speed and quality, often leading to the use of fast generic gradients (5-95% organic mobile phase) to purify compounds of various structures and polarity, which can be sometimes detrimental to the purity of the isolated compound. Isocratic elution is the best method to obtain high purity, but it usually requires a long trial and error method development to find the right eluent composition. Therefore it is mostly used to purify large quantities of product using for instance stack injections, but not for routine purification of structurally diverse compounds. Herein, we describe an empirical method that allows "right-first-time" purifications with isocratic elution on preparative RP-LCMS, permitting isolation of compounds in high purity and yield.

Orthogonal analytical screening for liquid chromatography-mass spectrometry method development and preparative scale-up.

An analytical HPLC-MS screening methodology has been developed to improve preparative RP-HPLC-MS purifications in medicinal chemistry laboratories. Although several approaches have been previously described to optimize analytical separations, none of them met our needs for the optimization of preparative conditions. Our screening protocol is based on searching among several orthogonal conditions to find the optimum preparative separation. Five different buffer conditions, from low to high pH, two organic solvents, acetonitrile and methanol, and five stationary phases of different polarities and characteristics were used. The orthogonality of the system was demonstrated using both, a standard mixture and mixtures from synthesis. To carry out the screening one of the analytical "open access" HPLC-MS systems was modified to perform the analytical screening while maintaining the open-access functionality for synthesis reaction monitoring. A software tool for automated sample programming and data reporting was also developed.

Gas chromatographic separation of isotopic molecules using a cavitand-impregnated ionic liquid stationary phase.

Cavitands, which are a class of compounds with deep open-ended cavities, are known to exhibit remarkable molecular recognition ability through host-guest interactions because of their unique structures. It is known that isotopic molecules can be differentiated in the small spaces provided by completely closed capsules in solution. To determine if this subtle molecular recognition ability extends to cavitands, we have developed a new method to facilitate the use of cavitands as stationary phases (SPs) in gas chromatography (GC). These involve a "pseudo heterogeneous two-phase system"; specifically, ionic liquids (ILs) were used as solvents to coat three cavitands with slightly different structures onto GC columns. When cavitand-coated columns were compared with columns coated with only ILs, it was clear that cavitands not only extended the utilization but also substantially improved separation efficiency of the columns. It was found that cavitand-coated columns can effectively separate many different types of isotopic compounds including aromatic hydrocarbons (mixture of chlorobenzene-h(5) and chlorobenzene-d(5), mixture of 1,2-dichlorobenzene-h(4) and 1,2-dichlorobenzene-d(4)), alcohols (methanol from its corresponding methanol-d, d(3), d(4)), ether, pyridine, and acetonitrile. The results also show that by modifying functional groups of the cavitand, from Et to C(11)H(23) or from amide to carboxylate, we can fully retain the molecular recognition ability of the cavitand. The drastic changes in the polarity of the SP from medium polar to nonpolar, or to polar, greatly extends the applicability of these cavitand-coated SPs. Compared to other GC SPs that are known to separate isotopic molecules, these cavitand-coated SPs can separate a relatively wider range of isotopic compounds at relatively lower temperature, with shorter column length and higher efficiency.

Angiotensin-converting enzyme 2 catalytic activity in human plasma is masked by an endogenous inhibitor.

Angiotensin-converting enzyme 2 (ACE2) is thought to act in an opposing manner to its homologue, angiotensin-converting enzyme (ACE), by inactivating the vasoconstrictor peptide angiotensin II and generating the vasodilatory fragment, angiotensin(1-7). Both ACE and ACE2 are membrane-bound ectoenzymes and may circulate in plasma as a consequence of a proteolytic shedding event. In this study, we show that ACE2 circulates in human plasma, but its activity is suppressed by the presence of an endogenous inhibitor. Partial purification of this inhibitor indicated that the inhibitor is small, hydrophilic and cationic, but not a divalent metal cation. These observations led us to develop a method for removal of the inhibitor, thus allowing detection of plasma ACE2 levels using a sensitive quenched fluorescent substrate-based assay. Using this technique, ACE2 activity measured in plasma from healthy volunteers (n = 18) ranged from 1.31 to 8.69 pmol substrate cleaved min-1 ml-1 (mean +/- s.e.m., 4.44 +/- 0.56 pmol min-1 ml-1). Future studies of patients with cardiovascular, renal and liver disease will determine whether plasma ACE2 is elevated in parallel with increased tissue levels observed in these conditions.

Comparison of an acetonitrile extraction/partitioning and "dispersive solid-phase extraction" method with classical multi-residue methods for the extraction of herbicide residues in barley samples.

An acetonitrile/partitioning extraction and "dispersive solid-phase extraction (SPE)" method that provides high quality results with a minimum number of steps and a low solvent and glassware consumption was published in 2003. This method, suitable for the analysis of multiple classes of pesticide residues in foods, has been given an acronymic name, QuEChERS, that reflects its major advantages (quick, easy, cheap, effective, rugged, safe). In this work, QuEChERS method, which was originally created for vegetable samples with a high amount of water, was modified to optimise the extraction of a wide range of herbicides in barley. Then, it was compared with known conventional multi-residue extraction procedures such as the Luke method, which was simplified and shortened by eliminating the Florisil clean-up (mini Luke) and the ethyl acetate extraction, which involves a subsequent clean-up by gel permeation chromatography (GPC) and which is the official extraction method used by some of European authorities. Finally, a simple acetone extraction was carried out to check the differences with the other three methods. Extracts were analysed by gas chromatography-time-of-flight mass spectrometry (GC-TOF/MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Mini Luke was significantly more effective for the extraction of non-polar and medium-polar compounds, but the best recoveries for polar compounds were achieved by QuEChERS and ethyl acetate methods. QuEChERS was the only method that provided an overall recovery value of 60-70% for non-, medium- and polar compounds, with some exceptions due to co-eluted matrix interferences. Clean-up by dispersive SPE was effective and did not differ so much with ethyl acetate extracts considering that QuEChERS clean-up step is much easier and less time-consuming. As a conclusion, it resulted to be the most universal extraction method by providing a well-defined phase separation without dilution and achieving acceptable recoveries in average including the extraction of the always difficult acidic herbicides. However, recoveries were not as good as required for validation purposes suggesting that residues are prone to strong matrix interactions in dry samples as barley and further method adaptation incrementing solvent strength, extraction time or more acidic or basic conditions is needed in order to achieve a complete extraction.