Low Level of Allergens in the Argentinean Plant Zuccagnia punctata Cav.: Screening and Quality Control of North-Western Propolis Using an LC-DAD-QTOF System

North-western Argentinean propolis (NAP), having promising bioactivity, was recently included into the National Food Code. Zuccagnia punctata Cav., a native shrub of north-western Argentina, is one of the prevalent botanical sources of NAPs, but no information on its allergenic constituents was available so far. A liquid chromatography-diode array detector -quadrupole-time of flight system (LC-DAD-QTOF) was used as a screening method for the reliable identification of sensitizing agents belonging to caffeic acid derivatives in Z. punctata and in two NAPs collected in the provinces of Catamarca and Tucum\ue1n. Caffeic acid phenethyl ester, one of the most active allergens in propolis, was never detected in either Z. punctata or NAP. Among 31 sensitizers, only geranyl caffeate was alleged in Z. punctata as <10% of its major constituent, whereas three caffeic acid derivatives with strong allergenic effect, i.e., geranyl, pentenyl, and benzyl caffeates, occurred in NAP samples (29%\u201336% of the Z. punctata major constituent), indicating other minor botanical sources. However, the high content of chalcones and flavonoids ascribed to Z. punctata significantly contributes to the antiallergenic and antioxidant character of these NAPs. This peculiar chemical profile depends on the extremophile condition in which this shrub grows and suggests other studies to characterize such raw materials for oral and topical formulation

In Vitro and In Vivo Human Herpesvirus 8 Infection of Placenta

Herpesvirus infection of placenta may be harmful in pregnancy leading to disorders in fetal growth, premature delivery, miscarriage, or major congenital abnormalities. Although a correlation between human herpesvirus 8 (HHV-8) infection and abortion or low birth weight in children has been suggested, and rare cases of in utero or perinatal HHV-8 transmission have been documented, no direct evidence of HHV-8 infection of placenta has yet been reported. The aim of this study was to evaluate the in vitro and in vivo susceptibility of placental cells to HHV-8 infection. Short-term infection assays were performed on placental chorionic villi isolated from term placentae. Qualitative and quantitative HHV-8 detection were performed by PCR and real-time PCR, and HHV-8 proteins were analyzed by immunohistochemistry. Term placenta samples from HHV-8-seropositive women were analyzed for the presence of HHV-8 DNA and antigens. In vitro infected histocultures showed increasing amounts of HHV-8 DNA in tissues and supernatants; cyto- and syncitiotrophoblasts, as well as endothelial cells, expressed latent and lytic viral antigens. Increased apoptotic phenomena were visualized by the terminal deoxynucleotidyl transferase-mediated deoxyuridine nick end-labeling method in infected histocultures. Ex vivo, HHV-8 DNA and a latent viral antigen were detected in placenta samples from HHV-8-seropositive women. These findings demonstrate that HHV-8, like other human herpesviruses, may infect placental cells in vitro and in vivo, thus providing evidence that this phenomenon might influence vertical transmission and pregnancy outcome in HHV-8-infected women

Analisi quantitativa delle dimetilarginine (ADMA ed SDMA) e degli aminoacidi strutturalmente correlati mediante UPLC-ESI-MS/MS nella diagnosi e prognosi di patologie NO-dipendenti correlate allo stress ossidativo nell'adulto e nel bambino.

Background: Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide (NO) formation inhibitor, has emerged as a promising biomarker of NO-associated endothelial dysfunction in cardiovascular diseases as well in chronic renal failure and pre-eclampsia but only recently this amino acid was associated also to the pathogenesis of some respiratory diseases, such asthma and cystic fibrosis. Although ADMA is an established marker of endothelial dysfunction in adults its biological relevance in the pediatric age is still unclear. Theoretically, due to its interference with nitric oxide (NO) production, ADMA might be involved in the pathogenesis of several disorders originating early in life. Interest has been specially focused on its potential role in neonatal lung biology, as NO is a critical regulator of lung morphogenesis and homeostasis. ADMA has been indicated as a mediator of the physiologically high pulmonary vascular resistance during fetal life, and implicated in the pathogenesis of persistent pulmonary hypertension of the newborn. Thus, there is considerable demand for a specific, sensitive and rapid method for ADMA and related metabolites for quantitative determination in biological fluids of adults with NO related disorders and preterm newborn involving in clinical study for diagnosis of cardiovascular and pulmonary diseases. The interest in potentially fundamental role of this metabolite, in basic and clinical research, led to the development of numerous analytical methods for the quantitative determination of ADMA and dimethylarginines in biological systems, notably plasma, serum and urine, but not, at the moment, in exhaled breath condensate (EBC), which is a potential rich source for countless biomarkers that can provide valuable information about respiratory as well as systemic diseases. EBC is obtained as breath exhaled from the lungs into a cooled collecting device, thereby condensing the vapor and aerosolized droplets emerging with the breath. Quantification of ADMA and related metabolites was made by several methods based on HPLC, coupled with fluorescence and mass spectrometric methods such as LCâMS/MS and GCâMS, capillary electrophoresis and ELISA. Moreover to apply the method in pediatric clinical research is very important to have a small volume of sample used for analysis, especially for plasma and serum. Objectives: The aim of this thesis was finalized to applied the method, created and validated in our laboratory, for simultaneous determination of arginine, ADMA, SDMA, NMMA, homo-arginine and citrulline in plasma samples of healthy adults and patients with different pathologies such as hyperglycemia, heart diseases and pre-eclampsia, and in plasma and serum of term and preterm newborns. To evaluate if ADMA is detectable in exhaled breath condensate of asthmatic and healthy children, we developed an alternative technique that can enhance our UPLC/MS/MS method in term of sensitivity using large injection volumes enriched online in a trap column that would compensate for the possible dilution effects in EBC samples. Methods: The method was characterized by simple and rapid preparation, short time analysis, accurate quantification on different biological matrices (plasma, serum and EBC) using stable isotope labeled internal standard and very low amount of sample by Ultrahigh-Performance Liquid Chromatography (UPLC) run coupled with electrospray ionization (ESI) in the positive mode tandem mass spectrometry detection. We measured circulating ADMA and related compounds concentrations in a group of very premature newborns at birth and during the first week of life. We enrolled different type of pathologic populations versus normal populations: adult control group was constituted of 36 healthy subject (18 males and 18 females, medium age 36.5 years) and 15 women in normal pregnancy (medium age of 32 years); and pathologic population was constituted of 15 women with pre-eclamptic pregnancy (medium age of 32 years), 20 subjects with heart diseases (10 males and 10 females, medium age of 75.5 years) and 30 hyperglycemic patients (16 males and 14 females, medium age 71 years). In pediatric field we enrolled a group of 31 extremely low birth weight (ELBW) newborns versus 30 term newborn to evaluate whether ADMA levels correlate with specific perinatal/neonatal risk factors or short-term outcome indicators; and EBC samples from a group of 29 children with asthma 5,8 to 16,3 years old and 62 healthy controls 5,1 to 15,4 years old. Results and conclusions: In healthy adults plasma and serum we did not found appreciable differences in concentration of ADMA and related metabolites, except for Arg and relative ratio versus ADMA, that appeared higher more than 60% in serum than in plasma. In adult subjects with heart diseases ADMA and SDMA values were respectively of 0.89±0.17µM and 0.72±0.26µM, significantly higher than normal controls (p<0.05); hyperglycemic subjects, instead have shown ADMA levels of 0.47±0.13µM significantly lower (p=0.002) than healthy subjects. There was any significant difference in SDMA concentrations between two studied populations. We observed that ADMA concentrations are significantly lower in pregnant pre-eclamptic women than in the control group at the time of delivery, but these data have some important limitation, owing to the small number of subjects enrolled and to the possible confounding effects of anti-hypertensive therapy in the pre-eclamptic group. In ELBW newborns ADMA, NMMA and SDMA concentrations remained stable from delivery to day 7 and were not dependent on gender, gestational age or birth weight. ADMA levels resulted significantly higher in infants born to mothers with histologic chorioamnionitis than in infants delivered for other maternal or fetal indications. We speculate that ADMA might be involved in the complex biological events associated with fetal exposure to chorioamnionitis. In this thesis, was also developed and validated a sensitive and selective method for the quantification of ADMA, arginine, SDMA, NMMA, citrulline, homo-arginine, tyrosine and nitrotyrosine simultaneously, in human EBC based on UPLCâMS/MS enrichment technology. Actually, do not exist in literature, any analytical method to detect ADMA and its related compounds in EBC human samples. The assay demonstrated excellent accuracy, precision, linearity and specificity for the intended clinical purpose and a good reproducibility of method of collection, demonstring that it is possible to measure ADMA in exhaled breath condensate. EBC ADMA values in asthmatic children (median value 2,17 [1,15-3,19] µmol/mL) were significantly higher than in healthy children (median value 1,1 [0,7-1,5] µmol/mL, p<0,001) and in the asthmatic group ADMA values were significantly correlated with inhaled corticosteroids dosage ICS (p=0,005; r=0,406). Conclusions: The UPLC-ESI/MS/MS method applied at adult populations with different NO-related endothelial dysfunctions, confirmed data from the literature. The fast run time, the feasibility of high sample throughput and the small amount of sample required make this method very suitable for routine analysis in the clinical setting. Preliminary data on neonatal and pediatric field, show that this method is a very good analytical tool for study dimethylarginines and their metabolism in healthy and disease conditions, it particularly suitable for future applications in the pediatric medicine. These data focus on ADMA as a biologically active molecule associated to exposure to antenatal inflammation, and we suggest its role deserves further investigation in larger clinical studies or appropriate experimental models. This was, also, the first study that demonstrates ADMA dosage feasibility in exhaled breath condensate, showing that ADMA is increased in asthma and has a possible role in asthmatic inflammation. Further studies will clarify the role of ADMA as a biomarker of asthmatic inflammation and a possible target for new pharmacological approaches.Presupposti: L' ADMA, dimetilarginina asimmetrica, è un analogo metilato dell'aminoacido arginina. Essa viene prodotta all'interno delle cellule attraverso processi di metilazione di catene polipeptidiche e rilasciata in circolo dopo proteolisi. L'ADMA compete con l'arginina, precursore necessario per la sintesi dell'ossido nitrico, per il legame con l'enzima ossido nitrico sintetasi (NOS), inibendone così la sintesi. LâNO è il più potente vasodilatatore conosciuto, che svolge un ruolo chiave, agendo sul sistema endoteliale, nel mantenimento di una grande varietà  di meccanismi omeostatici del nostro organismo. E' noto da tempo che l'ADMA, per inibizione della produzione di NO, è coinvolta nella patogenesi delle principali disfunzioni indotte da stress ossidativo NO-dipendente a livello cardiovascolare, renale e recentemente anche nell'ambito delle malattie respiratorie come l'asma e la fibrosi cistica, in quanto contribuisce ad aggravare lo stato infiammatorio e l'iperreattività  delle vie aeree. Analizzando i dati riportati in letteratura, si è notato che non vi sono studi longitudinali sulle concentrazioni di ADMA in età neonatale, sia nei soggetti a termine che in quelli prematuri, né sono stati ad oggi dosati i livelli di ADMA nel condensato dell'aria espirata, che si ritiene essere un biofluido la cui composizione riflette quella del liquido di superficie delle vie aeree, oltre ad essere caratterizzato dalla non invasività  e la semplicità  nell'esecuzione della metodica di prelievo. Scopo dello studio: il presente lavoro ha avuto in primo luogo lo scopo di applicare il metodo UPLC-ESI-MS/MS messo a punto nel nostro laboratorio, su una popolazione di adulti sani al fine di verificare la validità  della metodica, confermando i dati esistenti in letteratura. Si è poi passati all'applicazione su soggetti adulti affetti da diverse patologie (iperglicemia, cardiopatie, preeclampsia) per verificarne l'applicabilità  in ambito clinico, ed infine è stata presa in considerazione la popolazione pediatrica, determinando in maniera longitudinale la concentrazione sierica di ADMA e delle altre arginine metilate (SDMA, NMMA) in un gruppo di neonati a termine in prima e terza giornata di vita e in un gruppo di neonati pretermine di peso neonatale estremamente basso (< 1000 g - ELBW) durante il primo mese di vita. Per quanto riguarda lo studio delle malattie respiratorie lo scopo è stato quello di valutare se l'ADMA è dosabile nel condensato dell'aria espirata in bambini affetti da asma in buon controllo, con o senza terapia, confrontati con un gruppo di bambini sani e verificare la riproducibilità della metodica di analisi dell'ADMA sull'EBC. Materiali e metodi: La determinazione quantitativa è stata effettuata attraverso una separazione cromatografica in UPLC e analisi dei campioni mediante uno spettrometro di massa a triplo quadrupolo. Le caratteristiche del metodo sono costituite dall'utilizzo di quantità  esigue di campione, elevata specificità  e sensibilità  strumentale. Per poter ricavare i valori di riferimento della popolazione adulta sono stati arruolati 36 soggetti sani (18 uomini e 18 donne di età  media pari a 36.5 anni) come gruppo di controllo rispetto a pazienti con infarto miocardico (20 soggetti di cui 10 uomini e 10 donne di età  media pari a 75.5 anni) e diabete di tipo II insulino resistente (30 soggetti di cui 16 uomini e 14 donne di età  media pari a 71 anni). Lo studio sulle donne in gravidanza ha previsto l'arruolamento di 15 donne affette da pre-eclampsia confrontate con 15 donne in gravidanza normale (età  media 32 anni per entrambi i gruppi). L'applicazione in ambito pediatrico ha infine coinvolto un gruppo di 31 neonati di peso estremamente basso alla nascita (ELBW <1000 grammi) rispetto al gruppo di controllo di 30 neonati sani a termine, e un gruppo di 29 bambini asmatici di età compresa tra 5,8 e 16,3 anni confrontati con i relativi controlli sani (62 bambini di età  compresa tra 5,1 e 15,4 anni), di cui è stato raccolto il condensato dell'aria espirata. Risultati: I valori dei diversi metaboliti ottenuti sui soggetti adulti sani hanno confermato i dati riportati in letteratura, mentre nei soggetti cardiopatici le concentrazioni di ADMA ed SDMA sono state rispettivamente pari a 0.89±0.17µM e 0.72±0.26µM , risultando entrambi significativamente più alti rispetto ai controlli sani (p<0.05), a differenza invece dei soggetti iperglicemici che hanno mostrato livelli significativamente inferiori (p=0.002) di ADMA (0.47±0.13µM) rispetto ai relativi controlli, ma nessuna differenza nelle concentrazioni di SDMA. Il confronto tra donne gravide sane e con pre-eclampsia, ha invece evidenziato che la concentrazione di ADMA al momento del ricovero è stata significativamente più bassa nelle gravide ipertese rispetto alle gravide normotese (p=0.006), contrariamente a quanto riportato in letteratura, mentre a 30 giorni di distanza dal parto la situazione si è invertita. Nel gruppo dei neonati a termine, l'ADMA in prima giornata è risultata estremamente elevata, all'incirca 2-3 volte maggiore rispetto agli adulti. La sua concentrazione è diminuita rapidamente, riducendosi già  del 25% in terza giornata di vita. Nei soggetti pretermine di peso estremamente basso il dosaggio di ADMA è risultato sensibilmente più basso rispetto ai neonati a termine senza variazioni nel tempo. Considerando insieme i due gruppi di neonati, è presente una correlazione significativa tra valori di ADMA in prima ed in terza giornata e l'età  gestazionale, nonché il peso neonatale. Lo studio condotto sull'EBC ha, infine dimostrato che è possibile misurare l'ADMA nel condensato con una buona riproducibilità  intrasoggetto a distanza di 24 ore. I valori di ADMA nei bambini asmatici (mediana 2,17 [1,15-3,19] µmol/mL) sono risultati significativamente più elevati rispetto a quelli dei bambini sani (mediana 1,1 [0,7-1,5] µmol/mL, p<0,001) evidenziando anche una significativa correlazione dei livelli di questo metabolita con il dosaggio dei farmaci corticosteroidei inalatori ICS (p=0,005; r=0,406) assunti dai soggetti asmatici. Conclusioni: Il metodo sviluppato in questo studio soddisfa i requisiti analitici di precisione, riproducibilità , specificità , sensibilità  e recupero analitico indispensabili per l'impiego in ambito clinico. Esso inoltre può essere facilmente automatizzabile e adatto per il suo impiego su larga scala consentendo l'applicazione in qualità  di metodo diagnostico nell'ambito delle patologie da stress ossidativo e disfunzione endoteliale e delle malattie cardiovascolari. L'uso di una quantità  esigua di campione, inoltre ne suggerisce l'impiego per lo studio delle diverse patologie del neonato come la sepsi, l'ipertensione polmonare e lo scompenso cardiaco. Infine, essendo questo il primo studio che dimostra la fattibilità  del dosaggio dell'ADMA e dei biomarker di stress ossidativo nel condensato dell'aria espirata, ottenendo risultati concordanti con lavori precedenti che dimostravano un aumento di ADMA nell'asma in altre matrici biologiche, ci sono i presupposti per estendere la casistica per confermare il suo possibile ruolo come biomarker nell'infiammazione asmatica e come possibile target di nuovi approcci farmacologici

Quantification of Underivatised Amino Acids on Dry Blood Spot, Plasma, and Urine by HPLC\u2013ESI\u2013MS/MSAmino Acid Analysis

Enzyme deficiencies in amino acid (AA) metabolism affecting the levels of amino acids and their derivatives in physiological fluids may serve as diagnostically significant biomarkers for one or a group of metabolic disorders. Therefore, it is important to monitor a wide range of free amino acids simultaneously and to quantify them. This is time consuming if we use the classical methods and more than ever now that many laboratories have introduced Newborn Screening Programs for the semiquantitative analysis, detection, and quantification of some amino acids needed to be performed in a short time to reduce the rate of false positives.We have modified the stable isotope dilution HPLC-electrospray ionization (ESI)-MS/MS method previously described by Qu et al. (Anal Chem 74: 2034-2040, 2002) for a more rapid, robust, sensitive, and specific detection and quantification of underivatised amino acids. The modified method reduces the time of analysis to 10 min with very good reproducibility of retention times and a better separation of the metabolites and their isomers.The omission of the derivatization step allowed us to achieve some important advantages: fast and simple sample preparation and exclusion of artefacts and interferences. The use of this technique is highly sensitive, specific, and allows monitoring of 40 underivatized amino acids, including the key isomers and quantification of some of them, in order to cover many diagnostically important intermediates of metabolic pathways.We propose this HPLC-ESI-MS/MS method for underivatized amino acids as a support for the Newborn Screening as secondary test using the same dried blood spots for a more accurate and specific examination in case of suspected metabolic diseases. In this way, we avoid plasma collection from the patient as it normally occurs, reducing anxiety for the parents and further costs for analysis.The same method was validated and applied also to plasma and urine samples with good reproducibility, accuracy, and precision. The fast run time, feasibility of high sample throughput, and small amount of sample required make this method very suitable for routine analysis in the clinical setting

Use of a Mixed Cationic-Reverse Phase Column for Analyzing Small Highly Polar Metabolic Markers in Biological Fluids for Multiclass LC-HRMS Method

The determination of small highly polar metabolites at low concentrations is challenging when reverse-phase (RP) chromatography is used for multiclass analysis. A mixed cationic-RP column coupled to high-resolution tandem MS (HR-MS/MS) was tested for highly polar compounds in biological fluids, i.e., trimethylamine N-oxide (TMAO) and the isobaric molecules beta-methylamino-L-alanine (BMAA) and 2,4-diaminobutyric acid (DAB). The efficient retention and separation of the above compounds were obtained with common and MS-friendly RP conditions, reaching high selectivity and sensitivity. The method was firstly assessed in plasma and urine, showing good linearity in the range 50&ndash;1000 &micro;g/L and 500&ndash;10,000 &micro;g/L for TMAO and both BMAA and DAB, respectively. Excellent precision (RDS &lt; 3%) and good accuracies (71&ndash;85%) were observed except for BMAA in plasma, whose experimental conditions should be specifically optimized. Preliminary tests performed on compounds with biological relevance and a wider range of polarities proved the effectiveness of this chromatographic solution, allowing the simultaneous analysis of a larger panel of metabolites, from very small and polar compounds, like trimethylamine, to quite lipophilic molecules, such as corticosterone. The proposed LC-HRMS protocol is an excellent alternative to hydrophilic interaction liquid chromatography and ion-pairing RP chromatography, thus providing another friendly analytical tool for metabolomics

Simultaneous quantitative determination of NG,NG-dimethyl-l-arginine or asymmetric dimethylarginine and related pathway's metabolites in biological fluids by ultrahigh-performance liquid chromatography/electrospray ionization-tandem mass spectrometry

BACKGROUND: Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide (NO) formation inhibitor, has emerged as a promising biomarker of NO-associated endothelial dysfunction in cardiovascular diseases as well in chronic renal failure. The interest in potentially fundamental role of this metabolite, in basic and clinical research, led to the development of numerous analytical methods for the quantitative determination of ADMA and dimethylarginines in biological systems, notably plasma, serum and urine. OBJECTIVES: The aim of this work was to present a simple, fast and accurate UPLC-tandem-MS-based method for the simultaneous determination and quantification of arginine, ADMA, SDMA, NMMA, homo-arginine and citrulline. This method is designed for high sample throughput of only 10 \u3bcL of human plasma, serum or urine. METHODS: The analysis time is reduced to 1.9 min by an ultrahigh-performance liquid chromatography run coupled with electrospray ionization (ESI) in the positive mode tandem mass spectrometry detection. RESULTS: The method was validated in plasma, serum and urine. Correlation coefficients (r(2)) of the calibration curves in all matrices considered ranged from 0.9810 to 0.9993. Inter- and intra-assay precision, accuracy, recovery and carry-over were evaluated for validation. The LOD was 0.01 \u3bcM for all compounds in water, plasma and serum and 0.1 \u3bcM in urine. The LOQ was 0.05 \u3bcM for ADMA, SDMA, NMMA and H-Arg and 0.5 \u3bcM for Arg and Cit in water, plasma and serum; while in urine was 0.1 \u3bcM for ADMA, SDMA, NMMA and H-Arg and 0.5 \u3bcM for Arg and Cit. The precision was ranged from 1% to 15% expressed as CV% and the accuracy (bias %) was <\ub17% for all added concentrations with the exception of NMMA (-10%). ADMA mean plasma levels, measured in healthy adults and newborns, were in accord with literature data published: (M\ub1SD) 0.56\ub10.10 \u3bcM and 0.84\ub10.21 \u3bcM, respectively, showing that ADMA levels in plasma decreased with age. In serum we have similar data (0.54\ub10.18 \u3bcM and 1.14\ub10.36 \u3bcM), while in neonatal urine ADMA was 11.98\ub17.13 \u3bcmol mmol(-1) creatinine. CONCLUSIONS: Data from calibration curves and method validation reveal that the method is accurate and precise. The fast run time, the feasibility of high sample throughput and the small amount of sample required make this method very suitable for routine analysis in the clinical setting

Liquid chromatography-high resolution mass spectrometric methods for the surveillance monitoring of cyanotoxins in freshwaters

A comprehensive risk management on human exposure to cyanotoxins, whose production is actually unpredictable, is limited by reliable analytical tools for monitoring as many toxic algal metabolites as possible. Two analytical approaches based on a LC-QTOF system for target analysis and suspect screening of cyanotoxins in freshwater were presented. A database with 369 compounds belonging to cyanobacterial metabolites was developed and used for a retrospective data analysis based on high resolution mass spectrometry (HRMS). HRMS fragmentation of the suspect cyanotoxin precursor ions was subsequently performed for correctly identifying the specific variants. Alternatively, an automatic tandem HRMS analysis tailored for cyanotoxins was performed in a single chromatographic run, using the developed database as a preferred precursor ions list. Twenty-five extracts of surface and drinking waters contaminated by cyanobacteria were processed. The identification of seven uncommon microcystins (M(O)R, MC-FR, MSer7-YR, D-Asp3MSer7-LR, MSer7-LR, dmAdda-LR and dmAdda-YR) and 6 anabaenopeptins (A, B, F, MM850, MM864, oscyllamide Y) was reported. Several isobaric variants, fully separated by chromatography, were pointed out. The developed methods are proposed to be used by environmental and health agencies for strengthening the surveillance monitoring of cyanotoxins in wate

Use of a LC-DAD-QTOF system for the characterization of the phenolic profile of the argentinean plant Zuccagnia punctata and of the related propolis: New biomarkers

Argentinean northwestern propolis were included into the national Food Code, some of that were sometimes associated with Zuccagnia punctata, an endemic plant with interesting biological properties. An analytical protocol for characterizing the phenolic profile of Z. punctata by LC coupled to DAD-QTOF detection systems was proposed. Mass spectrometric and chromatographic selectivity allowed to recognize various compounds including isomers. Eleven compounds never mentioned before for this shrub were highlighted. Among them, two uncommon dihydrochalcones, i.e. 4'-hydroxy-2'-methoxydihydrochalcone and 2',4'-dihydroxydihydrochalcone, were described for the first time as major constituents of Z. punctata, suggesting a peculiar biosynthetic pathway. Only the two propolis collected in the \u201cDel Monte\u201d phytogeographical region showed large amounts of molecules present in Z. punctata, which were proposed as markers of propolis type Z. punctata. Since Z. punctata exhibits a very interesting bioactivity, propolis derived from this plant can be used as nutraceutical foo

Characterization of Lipopeptides Produced by Bacillus licheniformis using Liquid Chromatography with Accurate Tandem Mass Spectrometry

The plant endophyte Bacillus licheniformis, isolated from leaves of Vitis vinifera, was studied to individuate and characterize the presence of bioactive lipopeptides having aminoacidic structures