Performance Liquid Chromatography-mass Spectrometry Analysis Research Articles

MzmL, a novel marine derived N-acyl homoserine lactonase from Mesoflavibacter zeaxanthinifaciens that attenuates Pectobacterium carotovorum subsp. carotovorum virulence.

Quorum sensing (QS) is a conserved cell-cell communication mechanism widely distributed in bacteria, and is oftentimes tightly correlated with pathogen virulence. Quorum quenching enzymes, which interfere with QS through degrading the QS signaling molecules, could attenuate virulence instead of killing the pathogens, and thus are less likely to induce drug resistance. Many Gram-negative bacteria produce N-acyl homoserine lactones (AHLs) for interspecies communication. In this study, we isolated and identified a bacterial strain, Mesoflavibacter zeaxanthinifaciens XY-85, from an Onchidium sp. collected from the intertidal zone of Dapeng Reserve in Shenzhen, China, and found it had strong AHL degradative activity. Whole genome sequencing and blast analysis revealed that XY-85 harbors an AHL lactonase (designated MzmL), which is predicted to have an N-terminal signal peptide and share the "HXHXDH" motif with known AHL lactonases belonging to the Metallo-β-lactamase superfamily. Phylogenetic studies showed MzmL was closest to marine lactonase cluster members, MomL and Aii20J, instead of the AiiA type lactonases. Ultra performance liquid chromatography-mass spectrometry analysis confirmed that MzmL functions as an AHL lactonase catalyzing AHL degradation through lactone hydrolysis. MzmL could degrade both short- and long-chain AHLs with or without a substitution of oxo-group at the C-3 position, and retained full bioactivity under a wide range of temperatures (28-100°C) and pHs (4-11). Furthermore, MzmL significantly reduced Pectobacterium carotovorum subsp. carotovorum virulence factor production in vitro, such as biofilm formation and plant cell wall degrading enzyme production, and inhibited soft rot development on potato slices. These results demonstrated that MzmL may be a novel type of AHL lactonase with good environmental stability, and has great potential to be developed into a novel biological control agent for bacterial disease management.

Impact of lipase treatment on cuticle wax structure and anthocyanin metabolism in postharvest blueberries

AbstractIn this paper, we explored the influence of lipase on cuticle wax and anthocyanin metabolism in blueberries. The results demonstrated a progressive alteration in wax crystal structure, transitions from rod‐like to short rod‐like and flaky, and even disappearance of the cuticle wax. Gas chromatography–mass spectrometry analysis revealed a reduction in esters, aldehydes, long‐chain alkanes, and total wax content following lipase treatment of blueberry fruits. Additionally, ultra high performance liquid chromatography–mass spectrometry analysis showed significant decreases in peonidin, petunidin, cyanidin, and delphinidin, accompanied by an increase in malvidin content due to lipase treatment. Subsequent investigations uncovered that lipase treatment inhibited the activities of key anthocyanin synthetic enzymes, namely phenylalanine ammonia‐lyase (PAL), chalcone isomerase (CHI), and dihydroflavonol reductase (DFR). Moreover, lipase treatment downregulated the expression of synthesis‐related genes, including PAL, 4‐coumarate: CoA ligase, chalcone synthase (CHS), CHI, and DFR. In summary, our findings indicate that lipase disrupts the integrity of cuticle wax structure, inhibits anthocyanin synthesis, and reduces anthocyanin content. Accordingly, our studies suggest that inhibiting lipase could offer a novel approach to preserving the freshness and anthocyanin concentration of blueberries.

The Effect of Extraction Methods on Phytochemicals and Biological Activities of Green Coffee Beans Extracts.

The objectives of the present study consisted of identifying the impact of extraction methods and parameters held over the phytochemistry and biological activities of green coffee beans. Extraction processes belonging to two categories were performed: classical methods-maceration, Soxhlet extraction, and such innovative methods as turboextraction, ultrasound-assisted extraction, and a combination of the latter two. Total polyphenolic and flavonoid content, as well as in vitro antioxidant activity of the resulted extracts were spectrophotometrically determined. Extracts displaying the highest yields of bioactive compounds were subjected to High Performance Liquid Chromatography-Mass Spectrometry analysis. The extracts with the best phytochemical profiles were selected for biological activity assessment. In vivo, a model of plantar inflammation in Wistar rats was used to determine antioxidant activity, by evaluating the oxidative stress reduction potential, and anti-inflammatory activity. In vitro antimicrobial activity was also determined. The Soxhlet extraction and ultrasound-assisted extraction gave the highest bioactive compound yields. The highest total polyphenolic content was 2.691 mg/mL gallic acid equivalents and total flavonoid content was 0.487 mM quercetin equivalents for the Soxhlet extract subjected to 60 min extraction time. Regarding the antioxidant activity, ultrasound-assisted extraction reached the highest levels, i.e., 9.160 mg/mL Trolox equivalents in the DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) assay and 26.676 mM Trolox equivalents in the FRAP (Ferric Reducing Antioxidant Power) assay, at a 30 min extraction time and 50 °C extraction temperature. The 60 min Soxhlet extract reached the highest level for the ABTS+ (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) assay, 16.136 mM Trolox equivalents, respectively. Chlorogenic acid was present in the highest concentration in the same Soxhlet extract, 1657.179 µg/mL extract, respectively. Sterolic compounds were found in high concentrations throughout all the analyzed extracts. A proportional increase between yields and extraction parameter values was observed. Increased inhibition of Gram-negative bacteria was observed. The finally selected Soxhlet extract, that of 60 min extraction time, presented a significant in vivo antioxidant activity, with a slight anti-inflammatory activity. Antioxidant levels were elevated after 2 h of extract administration. Pro-inflammatory cytokine secretion was not influenced by the administration of the extract.

Expression of a Stilbene Synthase Gene from the Vitis labrusca x Vitis vinifera L. Hybrid Increases the Resistance of Transgenic Nicotiana tabacum L. Plants to Erwinia carotovora.

‘Isabel’ grape (Vitis labrusca x V. vinifera L. hybrid) is one of the main grape cultivars in Russia and some other countries for processing, due to its vigor, tolerance to the main fungal diseases, high yield and potential for sugar accumulation. The stilbene synthase gene VlvSTS was isolated from the hybrid grape cv. Isabel and cloned into a pSS plant transformation vector under the control of a constitutive 35S RNA double promoter of the cauliflower mosaic virus, CaMV 35SS. VlvSTS-gene containing transgenic tobacco lines were obtained and analyzed. For the first time plants expressing the VlvSTS gene were shown to have an enhanced resistance to the bacterial pathogen Erwinia carotovora subsp. carotovora B15. Transgenic plants were tested for resistance to a number of fungal pathogens. The plants were resistant to the grey mould fungus Botrytis cinerea, but not to the fungi Fusarium oxysporum, F. sporotrichioides, or F. culmorum. According to the results of a high performance liquid chromatography-mass spectrometry analysis, the amount of trans-resveratrol in leaves of transgenic plants with the highest expression of the VlvSTS gene was in a range from 150 to 170 μg/g of raw biomass. Change in the color and a decreased anthocyanin content in the flower corollas of transgenic plants were observed in transgenic lines with the highest expression of VlvSTS. A decrease in total flavonoid content was found in the flower petals but not the leaves of these tobacco lines. High expression of the VlvSTS gene influenced pollen development and seed productivity in transgenic plants. The size of pollen grains increased, while their total number per anther decreased. A decrease in the number of fertile pollen grains resulted in a decreased average weight of a seed boll in transgenic plants.

Ultra-high performance liquid chromatography-mass spectrometry analysis of plant apocarotenoids.

Apocarotenoids (APOs) are a class of carotenoid oxidation products with high structural and functional diversity. Apart from serving as precursors of phytohormones, fungal pheromones and vitamin A, several APOs act as signaling molecules involved in stress response and growth as regulators in plants. To comprehensively profile plant APOs, we established an improved ultra-high performance liquid chromatography-hybrid quadrupole-Orbitrap mass spectrometer (UHPLC-Q-Orbitrap MS) analytical platform. The improved APO analytical platform consists of an optimized sequential APO sample preparation and multiple UHPLC-MS detection methods and was successfully used to identify and quantify multiple subclasses of APOs from tomato fruits. By integrating ultrasound-assisted extraction, solid phase extraction, and chemical derivatization, the improved sequential APOs sample preparation facilitates the simultaneous preparation of different subclasses of APOs from plant materials. In addition, multiple UHPLC-MS detection methods enables high throughput analysis of APOs. Application of this analytical strategy can make important contributions to understanding the function of these compounds and significantly facilitate the elucidation of plant APO metabolism.

Triclosan degradation in sludge anaerobic fermentation and its impact on hydrogen production

Triclosan (TCS), a widely used antibacterial agent, was accumulated at significant levels in waste activated sludge (WAS). To date, however, the interaction between TCS and sludge anaerobic fermentation was rarely reported. Hence, this work aimed to deeply understanding the degradation of TCS in sludge anaerobic fermentation and its impact on hydrogen production. Experimental results showed that ~ 45% of TCS was degraded in long-term anaerobic fermentation, with 2,4-dichlorophenol as its main intermediate. Based on the information from high performance liquid chromatography–mass spectrometry analysis, three pathways i.e., dechlorination, hydroxylation, and cleavage of ether bonds, were proposed for TCS degradation. It was found that the maximum hydrogen yield decreased from 18.6 to 12.8 mL/g VSS with the increase of TCS from 12 to 487 mg/kg TSS. One possible reason for the decreased hydrogen yield was that a part of hydrogen generated might serve as electron donors for TCS dechlorination. Besides, the presence of TCS significantly suppressed acidogenesis (an important step responsible for hydrogen generation). This inhibition to acidogenesis is likely due to that the high-affinity functional groups of TCS such as hydroxyl groups could bind to the active sites of acetate kinase (AK, a key enzyme in acidogenesis), which reduced the active sites available for original fermentation substrates. Microbial analysis revealed that TCS increased the relative abundances of potential contaminant decomposers such as Guggeheimella but inhibited the populations of hydrogen producers such as Proteiniborus, which was consistent with the results obtained by chemical analyses.

Monitoring chemical synthesis of human atrial natriuretic peptide by ultra high performance liquid chromatography-mass spectrometry analysis

Peptides participate in and regulate many important life processes as neurotransmitters, hormones and receptors. With detailed understanding of the structure and function of peptides, chemically synthesized peptides have shown great value in the fields of biomedicine and life analysis. Human atrial natriuretic peptide (ANP) is a polypeptide hormone consisting of 28 amino acids. The abnormalities in the content and metabolism of ANP are closely related to the diseases like cardiovascular diseases and cancer. In view of the important physiological functions of ANP, herein we monitored and optimized the chemical synthesis of ANP chemical synthesis. The strategy involved integrating solid phase synthesis of linear peptide chain and liquid phase oxidation. Based on the excellent separation performance of HPLC and the excellent qualitative ability of mass spectrometry (MS), a new method based on ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was established to monitor the synthesis reaction and to separate and purify the obtained products. This study provides a reference for the efficient chemical synthesis and oxidative ring formation of human atrial natriuretic peptide.

Determination of ultraviolet filters in human nails using an acid sample digestion followed by ultra-high performance liquid chromatography–mass spectrometry analysis

Ultraviolet filters (UV-filters) are specific chemicals that absorb and reflect UVA and UVB radiation from the sun. They are regularly used in sunscreens and in other personal care products (PCPs), and in products like plastics, adhesives, toys, or furniture finishes. This work develops and validates a new method to determine concentrations of UV-filters (BP-1, BP-2, BP-3, BP-6, BP-8, 4-OH-BP, THB, AVB) in human nail samples. Nails are easily available and are considered to be suitable indicators of cumulative and continued exposure to harmful chemicals. The treatment of nail samples includes microwave assisted digestion/extraction (MAE) in a methanolic solution of o-phosphoric acid (0.05 mol L−1) followed by analyte determination using ultra-high performance liquid chromatography–mass spectrometry (UHPLC–MS/MS) in multiple reaction monitoring mode. The analytes were separated in less than 10 min. The digestion procedure was optimized using multivariate techniques. Matrix-matched calibration with a pig hoof matrix was used for validating the method. A study of accuracy with spiked blank samples was also conducted. The calculated detection limits varied between 0.2 and 1.5 ng g−1, and quantification limits between 1.0 and 5.0 ng g−1. The trueness of the method was an estimation of the recovery, which was between 90.2% and 112.2%; with an estimated precision (relative standard deviation, % RSD) lower than 12.3% for all UV-filters. Nail samples were obtained from 22 volunteers (male and female). The results showed that BP-1 and BP-3 mainly bioaccumulate in human nails.

Genome-based analysis for the bioactive potential of Streptomyces yeochonensis CN732, an acidophilic filamentous soil actinobacterium

BackgroundAcidophilic members of the genus Streptomyces can be a good source for novel secondary metabolites and degradative enzymes of biopolymers. In this study, a genome-based approach on Streptomyces yeochonensis CN732, a representative neutrotolerant acidophilic streptomycete, was employed to examine the biosynthetic as well as enzymatic potential, and also presence of any genetic tools for adaptation in acidic environment.ResultsA high quality draft genome (7.8 Mb) of S. yeochonensis CN732 was obtained with a G + C content of 73.53% and 6549 protein coding genes. The in silico analysis predicted presence of multiple biosynthetic gene clusters (BGCs), which showed similarity with those for antimicrobial, anticancer or antiparasitic compounds. However, the low levels of similarity with known BGCs for most cases suggested novelty of the metabolites from those predicted gene clusters. The production of various novel metabolites was also confirmed from the combined high performance liquid chromatography-mass spectrometry analysis. Through comparative genome analysis with related Streptomyces species, genes specific to strain CN732 and also those specific to neutrotolerant acidophilic species could be identified, which showed that genes for metabolism in diverse environment were enriched among acidophilic species. In addition, the presence of strain specific genes for carbohydrate active enzymes (CAZyme) along with many other singletons indicated uniqueness of the genetic makeup of strain CN732. The presence of cysteine transpeptidases (sortases) among the BGCs was also observed from this study, which implies their putative roles in the biosynthesis of secondary metabolites.ConclusionsThis study highlights the bioactive potential of strain CN732, an acidophilic streptomycete with regard to secondary metabolite production and biodegradation potential using genomics based approach. The comparative genome analysis revealed genes specific to CN732 and also those among acidophilic species, which could give some insights into the adaptation of microbial life in acidic environment.

Biomarker screening for antenatal depression in women who underwent caesarean section: a matched observational study with plasma Lipidomics

BackgroundAntenatal depression is a prevalent mental disorder in women who have undergone caesarean section, and it often presages adverse postoperative outcomes. Because of the lack of a laboratory-based diagnostic strategy, antenatal depression is mainly determined by a psychologist’s subjective judgment based on a structured clinical interview for established diagnostic criteria. However, the diagnostic accuracy rate for depression by non-psychiatrists is relatively low. Thus, this study aimed to use lipidomics to identify potential biomarkers related to antenatal depression in women who have undergone caesarean section.MethodsThe study was designed as a matched prospective observational study. Singleton pregnant women scheduled to receive elective caesarean section, were screened for eligibility. Women diagnosed with major antenatal depression were matched with non-antenatal depression controls in terms of age (±1 year) and BMI (±1 kg/m2), and blood samples of the included matched pairs were collected. Subsequently, lipidomics of the plasma samples were performed using Ultra Performance Liquid Chromatography-mass spectrometry analysis to explore the differentially expressed lipids in women with or without antenatal depression.ResultsIn total, 484 pregnant women were screened; 66 subjects were recruited, including 33 subjects with major antenatal depression and 33 matched controls without antenatal depression. Thirty-five differentially expressed lipid metabolites were identified (P < 0.05). The area under the receiver operating characteristic curve of these lipid metabolites was 0.7 or larger; the area under curve for cholesterol sulfate was 0.823 (95% CI: 0.716–0.930), and that of PC (18:2 (2E, 4E)/0:0) was 0.778 (95%CI: 0.662–0.895). In the conditional logistic stepwise regression analysis, cholesterol sulfate (P = 0.009) and PC (18:2 (2E, 4E)/0:0) (P = 0.035) were also identified as effective predictive risk factors for antenatal depression.ConclusionsWomen who had undergone caesarean section and experienced antenatal depression presented a significantly differentially expressed profile of plasma lipidomics compared to those who did not experience antenatal depression. Cholesterol sulfate and PC (18:2 (2E, 4E)/0:0) may be effective and specific lipidic biomarkers for the prediction of antenatal depression.Trial registrationChina Clinical Trial Registration Center registration number: ChiCTR1800016230; date of registration: 21/05/2018.

High Performance Liquid Chromatography-mass Spectrometry Analysis of High Antioxidant Australian Fruits with Antiproliferative Activity Against Cancer Cells.

Background:High antioxidant capacities have been linked to the treatment and prevention of several cancers. Recent reports have identified several native Australian fruits with high antioxidant capacities. Despite this, several of these species are yet to be tested for anticancer activity.Materials and Methods:Solvent extracts prepared from high antioxidant native Australian fruits were analyzed for antioxidant capacity by the di (phenyl)-(2,4,6-trinitrophenyl) iminoazanium free radical scavenging assay. Antiproliferative activities against CaCo2 and HeLa cancer cells were determined by a multicellular tumor spheroid-based cell proliferation assay. Toxicity was determined by Artemia franciscana bioassay.Results:Methanolic extracts of all plant species displayed high antioxidant contents (equivalent to approximately 7–16 mg of vitamin C per gram of fruit extracted). Most aqueous extracts also contained relatively high antioxidant capacities. In contrast, the ethyl acetate, chloroform, and hexane extracts of most species (except lemon aspen and bush tomato) had lower antioxidant contents (below 1.5 mg of vitamin C equivalents per gram of plant material extracted). The antioxidant contents correlated with the ability of the extracts to inhibit proliferation of CaCo2 and HeLa cancer cell lines. The high antioxidant methanolic extracts of all species were potent inhibitors of cell proliferation. The methanolic lemon aspen extract was particularly effective, with IC50 values of 480 and 769 μg/mL against HeLa and CaCo2 cells, respectively. In contrast, the lower antioxidant ethyl acetate and hexane extracts (except the lemon aspen ethyl acetate extract) generally did not inhibit cancer cell proliferation or inhibited to only a minor degree. Indeed, most of the ethyl acetate and hexane extracts induced potent cell proliferation. The native tamarind ethyl acetate extract displayed low-moderate toxicity in the A. franciscana bioassay (LC50 values below 1000 μg/mL). All other extracts were nontoxic. A total of 145 unique mass signals were detected in the lemon aspen methanolic and aqueous extracts by nonbiased high-performance liquid chromatography-mass spectrometry analysis. Of these, 20 compounds were identified as being of particular interest due to their reported antioxidant and/or anticancer activities.Conclusions:The lack of toxicity and antiproliferative activity of the high antioxidant plant extracts against HeLa and CaCo2 cancer cell lines indicates their potential in the treatment and prevention of some cancers.SUMMARY Australian fruit extracts with high antioxidant contents were potent inhibitors of CaCo2 and HeLa carcinoma cell proliferationMethanolic lemon aspen extract was particularly potent, with IC50 values of 480 μg/mL (HeLa) and 769 μg/mL (CaCo2)High-performance liquid chromatography-mass spectrometry-quadrupole time-of-flight analysis highlighted and putatively identified 20 compounds in the antiproliferative lemon aspen extractsIn contrast, lower antioxidant content extracts stimulated carcinoma cell proliferationAll extracts with antiproliferative activity were nontoxic in the Artemia nauplii assay. Abbreviations used: DPPH: di (phenyl)- (2,4,6-trinitrophenyl) iminoazanium, HPLC: High-performance liquid chromatography, IC50: The concentration required to inhibit by 50%, LC50: The concentration required to achieve 50% mortality, MS: Mass spectrometry.

High performance liquid chromatography-mass spectrometry analysis of high antioxidant Australian fruits with antiproliferative activity against cancer cells

High performance liquid chromatography-mass spectrometry analysis of high antioxidant Australian fruits with antiproliferative activity against cancer cells

Development and application of an analytical procedure for specific migration of green tea compounds in IV gamma nectarine active packaging

The aim of study was to develop an easy and reliable method for the quantification of tea compounds migrated from an active packaging to the foodstuff, being IV gamma nectarine the product tested. The method was based on a solid phase extraction (SPE) followed by ultra performance liquid chromatography-mass spectrometry analysis. SPE conditions were first optimized by spiking the nectarine flesh 1% of tea extract in methanol. A previous dilution of the sample extract in water (1:10) was required to remove the major matrix interferences. Then, the washing was optimized for 10 mL water and the extraction was optimized for 0.8 mL methanol. SPE method provided recoveries ranging between 81 and 99%. Limits of detection (0.006–0.048 μg kg−1), quantification (0.008–0.160 μg kg−1) and reproducibility below to 4.2% were obtained. The method has been applied to IV gamma nectarine packaging containing green tea extract. Samples were analyzed by the methodology described and the specific migration values of tea compounds were for all the cases below to the limit of detection calculated ensuring food safety and quality of nectarine active packaging.

Review article: Herbal hepatotoxicity--an update on traditional Chinese medicine preparations.

Although evidence for their therapeutic efficacy is limited, herbal traditional Chinese medicine (TCM) preparations increasingly gain popularity. In contrast to other herbal products, adverse effects by herbal TCM including liver toxicity were rarely reported. In recent years, more cases were published, providing new clinical challenges. To summarise comprehensively the literature on herbal TCM hepatotoxicity since 2011. PubMed was searched using key words related to TCM, the results were restricted to full English-language publications and abstracts published since 2011. In addition, the database of the National Institutes of Health (NIH) and LiverTox was accessed under the topic 'Drug record: Chinese and other Asian herbal medicines'. Since 2011, new case reports and case series provided evidence for herbal hepatotoxicity by TCM, focusing on nine TCM herbal mixtures and four individual TCM herbs with potential health hazards. These were the TCM products Ban Tu Wan, Chai Hu, Du Huo, Huang Qin, Jia Wei Xia Yao San, Jiguja, Kamishoyosan, Long Dan Xie Gan Tang, Lu Cha, Polygonum multiflorum products, Shan Chi, 'White flood' containing the herbal TCM Wu Zhu Yu and Qian Ceng Ta, and Xiao Chai Hu Tang. Other developments include the establishment of a new and early diagnostic serum marker for hepatotoxicity caused by pyrrolizidine alkaloids, assessed using ultra performance liquid chromatography-mass spectrometry analysis, and new regulatory details to improve herbal TCM product quality and safety. Stringent evaluation of the risk/benefit ratio is essential to protect traditional Chinese medicines users from health hazards including liver injury.

Preparation of flavonoid reference standards from &lt;I&gt;Scutellariae&lt;/I&gt; Radix under the guidance of high performance liquid chromatography-mass spectrometry analysis

Flavonoid reference standards were targeted-prepared from Scutellariae Radix under the guidance of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis. With HPLC-MS analysis of Scutellariae Radix, 19 flavonoid components were identified by analyzing and comparing their retention times, ultraviolet spectra, and mass spectrometry data with literature. The separation and purification protocols of all targeted flavonoid reference standards were optimally designed according to the results of HPLC-MS analysis and related literature. The ethanol extract of Scutellariae Radix was suspended in water and extracted with petroleum ether, ethyl acetate, and n-butanol successively. The ethyl acetate extract and n-butanol extract were separately subjected to primary separation by low pressure reverse phase preparative chromatography. Then the fractions containing targeted compounds were further purified by low pressure reverse and normal phases preparative chromatography. Finally, baicalin and wogonoside reference standards were obtained from n-butanol extract; baicaelin, wogonin, and oroxylin A reference standards were obtained from ethyl acetate extract. The structures of the 5 reference standards were identified by mass spectrometry (MS) and 1H nuclear magnetic resonance (1H NMR) spectroscopy. The HPLC analytical results showed that the purities of the 5 reference standards were all above 98%. It is demonstrated that the rapid targeted-preparation method under the guidance of the HPLC-MS analysis is applicable for the isolation and preparation of chemical components in traditional Chinese medicines.

Preparation of flavonoid reference standards from &lt;I&gt;Scutellariae&lt;/I&gt; Radix under the guidance of high performance liquid chromatography-mass spectrometry analysis

Preparation of flavonoid reference standards from &lt;I&gt;Scutellariae&lt;/I&gt; Radix under the guidance of high performance liquid chromatography-mass spectrometry analysis

Acute paralysis after seafood ingestion

The first European case of tetrodotoxin intoxication is reported in a patient who ingested a trumpet shellfish from the Atlantic Ocean in Southern Europe. He suffered general acute paralysis with respiratory failure necessitating ventilation. Early neurophysiologic studies showed complete peripheral nerve inexcitability, with no recordable sensory or motor responses, and normal electroencephalography. Tetrodotoxin was detected in high quantities in the patient's blood and urine through high performance liquid chromatography-mass spectrometry analysis. Seventy-two hours after admission the patient recovered normal strength, reflexes and sensation.

Occurrence of deoxynivalenol and deoxynivalenol-3-glucoside in durum wheat

The occurrence of deoxynivalenol and deoxynivalenol-3-glucoside in durum wheat samples (n=150; 25 lines × 2 reps × 3 environments) collected in 2010 from 3 areas located in north-central Italy was evaluated. In addition, the co-occurrence of other trichothecenes was considered. An optimised extraction method based on the use of salts followed by ultra-high performance liquid chromatography-mass spectrometry analysis was used for the quantification of the mycotoxins. All samples were found positive for deoxynivalenol at concentrations ranging between 47 and 3,715 μg/kg. A ubiquitous occurrence of deoxynivalenol-3-glucoside was found; 85% of the analysed samples contained this masked mycotoxin at concentrations varying between 46 and 842 μg/kg. In addition to glycosylated deoxynivalenol, acetylated forms of deoxynivalenol (3- and 15-acetyldeoxynivalenol) were also found in most of the durum wheat samples. The deoxynivalenol-3-glucoside/deoxynivalenol ratio, reaching up to 30% in many samples, was similar to that already found in other cereals such as soft wheat and barley. These data open the way for further investigations on the role of glycosylating activity as a possible Fusarium head blight-resistance mechanism in durum wheat, as already proved in the case of soft wheat.

Microbial Community Structure at Different Fermentation Stages of Kutajarista, a Herbal Formulation

Kutajarista is an Ayurvedic fermented herbal formulation prescribed for gastrointestinal disorders. This herbal formulation undergoes a gradual fermentative process and takes around 2months for production. In this study, microbial composition at initial stages of fermentation of Kutajarista was assessed by culture independent 16S rRNA gene clone library approach. Physicochemical changes were also compared at these stages of fermentation. High performance liquid chromatography-mass spectrometry analysis showed that Gallic acid, Ellagic acid, and its derivatives were the major chemical constituents recovered in this process. At 0day of fermentation, Lactobacillus sp., Acinetobacter sp., Alcaligenes sp., and Methylobacterium sp. were recovered, but were not detected at 8day of fermentation. Initially, microbial diversity increased after 8days of fermentation with 11 operational taxonomic units (OTUs), which further decreased to 3 OTUs at 30day of fermentation. Aeromonas sp., Pseudomonas sp., and Klebsiella sp. dominated till 30day of fermentation. Predominance of γ- Proteobacteria and presence of gallolyl derivatives at the saturation stage of fermentation implies tannin degrading potential of these microbes. This is the first study to highlight the microbial role in an Ayurvedic herbal product fermentation.

Antioxidant capacity and total phenolic contents of oregano (Origanum vulgare), lavender (Lavandula angustifolia) and lemon balm (Melissa officinalis) from Romania

The study reported here presents a comparative screening of three medicinal plants including oregano (Origanum vulgare L.), lavender (Lavandula angustifolia) and lemon balm (Melissa officinalis) having the same geographical origin, the Southeast region of Romania, and growing in the same natural conditions. The contents of total phenolics and total flavonoids for the extracts of these were determined. Furthermore, the total antioxidant capacity was also evaluated. It was found that Origanum vulgare and Melissa officinalis extracts present the most effective antioxidant capacity in scavenging DPPH radicals, while Lavandula angustifolia is less active. High performance liquid chromatography–mass spectrometry analysis was used to identify the components of extracts. Major phenolic acids identified in the analysed species were ferulic, rosmarinic, p-coumaric and caffeic, while predominant flavonoids were quercetin, apigenin kaempherol, which were present as glucosides.