The toxicological spectrum of the Bacillus cereus toxin cereulide points towards niche-specific specialisation.

Most microbes share their environmental niches with very different forms of life thereby engaging in specialised relationships to enable their persistence. The bacterium Bacillus cereus occurs ubiquitously in the environment with certain strain backgrounds causing foodborne and opportunistic infections in humans. The emetic lineage of B. cereus is capable of producing the toxin cereulide, which evokes emetic illnesses. Although food products favouring the accumulation of cereulide are known, the ecological role of cereulide and the environmental niche of emetic B. cereus remain elusive. To better understand the ecology of cereulide-producing B. cereus, we systematically assayed the toxicological spectrum of cereulide on a variety of organisms belonging to different kingdoms. As cereulide is a potassium ionophore, we further tested the effect of environmental potassium levels on the action of cereulide. We found that adverse effects of cereulide exposure are species-specific, which can be exacerbated with increased environmental potassium. Additionally, we demonstrate that cereulide is produced within an insect cadaver indicating its potential ecological function for a saprophytic lifestyle. Collectively, distinct cereulide susceptibilities of other organisms may reflect its role in enabling competitive niche specialization of emetic B. cereus.

(Bio)active Compounds in Daisy Flower (Bellis perennis).

The common daisy (Bellis perennis) belongs to the family Asteraceae and, in recent years, some new research has been published on the bioactive compounds and biological activities of its extracts. In 2014, the knowledge was partially summarized, but several new studies have been published in the last nine years. In addition, the substances were tabularly consolidated to give a comprehensive overview of over 310 individual components, compound classes, and bioactivities, as well as their accurate plant organ origin. The latest results have shown that the plant has antioxidative, antimicrobial, anticancerogenic, wound healing, antidepressive, anxiolytic, nephroprotective, and insulin mimetic effects, as well as an effect on lipid metabolism. Some studies in the field of homeopathy were also listed. Ideally, a biological effect and one or several compound(s) can be correlated. However, the compounds of the extracts used have often been qualified and quantified, but it remains unclear which of these substances have an activity. The works often stick at the level of the crude extract or a fraction, but not at a single purified and tested compound and, consequently, they are hampered by a missing comprehensive bioactivity workflow. This review provides a critical overview and gaps and offers a basis for further research in this area.

Rotenoids and Isoflavones from Xeroderris stuhlmannii (Taub.) Mendonça & E.P. Souza and Their Biological Activities.

The phytochemical study of the ethanolic extract of the leaf of Xeroderris stuhlmannii led to the isolation of five hitherto unreported compounds including two isoflavones (1-2), and three rotenoids (3-5), along with eight known isoflavonoid derivatives (6-13) and one pterocarpan derivative (14). The structures of the new compounds and those of the known ones were established by the spectroscopic (1D and 2D NMR) and spectrometric (HRESIMS) techniques as well as a comparison of their spectroscopic data with those reported in the literature. The leaf extract, fractions, and isolated compounds were tested for their antibacterial effects against nine bacterial strains. Compounds 3, 8, 11, and 12 showed a significant antibacterial effect, with a minimum inhibitory concentration (MIC) value of 62.5 µg/mL each, against Salmonella typhi, Staphylococcus aureus, Klessiella pneumonae, and Escherichia coli, respectively. In addition, the leaf extract, fractions, and isolated compounds were tested for their antifungal effects against four fungal strains. The hexane fraction showed a significant antifungal effect with an MIC value of 125 µg/mL against Candida parasilosis, whereas compounds 3, 8, and 12 showed significant antifungal activity with an MIC value of 62.5 µg/mL, each against Candida parasilosis, Candida albicans, and Candida krusei, respectively.

Impact of a Novel PagR-like Transcriptional Regulator on Cereulide Toxin Synthesis in Emetic Bacillus cereus.

The emetic type of foodborne disease caused by Bacillus cereus is produced by the small peptide toxin cereulide. The genetic locus encoding the Ces nonribosomal peptide synthetase (CesNRPS) multienzyme machinery is located on a 270 kb megaplasmid, designated pCER270, which shares its backbone with the Bacillus anthracis toxin plasmid pXO1. Although the ces genes are plasmid-borne, the chromosomally encoded pleiotropic transcriptional factors CodY and AbrB are key players in the control of ces transcription. Since these proteins only repress cereulide synthesis during earlier growth phases, other factors must be involved in the strict control of ces expression and its embedment in the bacterial life cycle. In silico genome analysis revealed that pCER270 carries a putative ArsR/SmtB family transcription factor showing high homology to PagR from B. anthracis. As PagR plays a crucial role in the regulation of the protective antigen gene pagA, which forms part of anthrax toxin, we used a gene-inactivation approach, combined with electrophoretic mobility shift assays and a bacterial two-hybrid system for dissecting the role of the PagR homologue PagRBc in the regulation of cereulide synthesis. Our results highlight that the plasmid-encoded transcriptional regulator PagRBc plays an important role in the complex and multilayered process of cereulide synthesis.

Bacillus cereus Toxin Repertoire: Diversity of (Iso)cereulide(s).

The emetic Bacillus cereus toxin cereulide (1) poses a significant safety risk in the food industry, causing emesis and nausea after consumption of contaminated foods. Analogously to cereulide, the structures of various isocereulides, namely, isocereulides A-G, have been recently reported and could also be identified in B. cereus-contaminated food samples. The HPLC fractionation of B. cereus extracts allows us to isolate additional isocereulides. By applying MSn sequencing, post-hydrolytic dipeptide, amino acid and α-hydroxy acid analyses using UPLC-ESI-TOF-MS to purify the analytes, seven new isocereulides H-N (2-8) could be elucidated in their chemical structures. The structure elucidation was supported by one-dimensional and two-dimensional NMR spectra of the isocereulides H (2), K (5), L and N (6 + 8) and M (7). The toxicity of 2-8 was investigated in a HEp-2 cell assay to determine their respective 50% effective concentration (EC50). Thus, 2-8 exhibited EC50 values ranging from a 0.4- to 1.4-fold value compared to cereulide (1). Missing structure-activity correlations indicate the necessity to determine the toxic potential of all naturally present isocereulides as single compounds to be able to perform a thorough toxicity evaluation of B. cereus-contaminated foods in the future.

Structure Revision of Isocereulide A, an Isoform of the Food Poisoning Emetic Bacillus cereus Toxin Cereulide.

The emetic Bacillus cereus toxin cereulide presents an enormous safety hazard in the food industry, inducing emesis and nausea after the consumption of contaminated foods. Additional to cereulide itself, seven structurally related isoforms, namely the isocereulides A-G, have already been elucidated in their chemical structure and could further be identified in B. cereus contaminated food samples. The newly performed isolation of isocereulide A allowed, for the first time, 1D- and 2D-NMR spectroscopy of a biosynthetically produced isocereulide, revealing results that contradict previous assumptions of an l-O-Leu moiety within its chemical structure. By furthermore applying posthydrolytical dipeptide analysis, amino acid and α-hydroxy acid analysis by means of UPLC-ESI-TOF-MS, as well as MSn sequencing, the structure of previously reported isocereulide A could be corrected. Instead of the l-O-Leu as assumed to date, one l-O-Ile unit could be verified in the cyclic dodecadepsipeptide, revising the structure of isocereulide A to [(d-O-Leu-d-Ala-l-O-Val-l-Val)2(d-O-Leu-d-Ala-l-O-Ile-l-Val)].

Phytochemical Characterization of Low Molecular Weight Constituents from Marshmallow Roots (Althaea officinalis) and Inhibiting Effects of the Aqueous Extract on Human Hyaluronidase-1.

Extract RE was obtained from the roots of Althaea officinalis in a yield of 8.1%, related to the dried plant material, by extraction with MeOH-H2O (1:1), followed by precipitation with EtOH to remove high molecular weight constituents. Phytochemical investigation of RE revealed the presence of N-phenylpropenoyl-l-amino acid amides 1-5, 8% glycine betaine 6, about 9% total amino acids with proline as the main compound, and about 61% mono- and oligomeric carbohydrates with sucrose as the main compound. Further fractionation revealed the presence of a hypolaetin diglycoside (12) and four hypolaetin glycosides (7-9 and 11) with O-sulfocarbohydrate moieties; additionally, 4'-O-methylisoscutellarein-8-O-ß-d-(3″-O-sulfo)glucuronopyranoside (10) and the diglycosylated coumarin haploperoside D (13) were identified. The hypolaetin-O-sulfoglycosides 7-10 are new natural products. RE inhibited the enzymatic activity of surface-displayed human hyaluronidase-1 on Escherichia coli F470 cells with an IC50 of 7.7 mg/mL. RE downregulated mRNA expression of hyal-1 in HaCaT keratinocytes at 125 and 250 µg/mL, respectively. These data contribute to a deeper phytochemical understanding of marshmallow root extracts and to the positive influence of extracts used for therapy of irritated and inflamed buccal tissue and cough.

Dual effectiveness of Alternaria but not Fusarium mycotoxins against human topoisomerase II and bacterial gyrase.

Type II DNA-topoisomerases (topo II) play a crucial role in the maintenance of DNA topology. Previously, fungi of the Alternaria genus were found to produce mycotoxins that target human topo II. These results implied the question why a fungus should produce secondary metabolites that target a human enzyme. In the current work, the homology between human topo II and its bacterial equivalent, gyrase, served as basis to study a potential dual inhibition of both enzymes by mycotoxins. A total of 15 secondary metabolites produced by fungi of the genera Alternaria and Fusarium were assessed for their impact on topo II of human and bacterial origin in the decatenation and the supercoiling assay, respectively. In line with the theory of dual topo II inhibition, six of the tested Alternaria mycotoxins were active against both enzymes, the dibenzo-α-pyrones alternariol (AOH) and alternariol monomethyl ether (AME), as well as the perylene-quinones altertoxin I (ATX I) and II (ATX II), alterperylenol (ALP) and stemphyltoxin III (STTX III). The Alternaria metabolites altersetin (ALN), macrosporin (MAC), altenusine (ALS) and pyrenophorol (PYR) impaired the function of human topo II, but did not show any effect on gyrase. The potency to inhibit topo II activity declined in the row STTX III (initial inhibitory concentration 10 µM) > AOH (25 µM) = AME (25 µM) = ALS (25 µM) = ATX II (25 µM) > ALN (50 µM) = ATX I (50 µM) > ALP (75 µM) = PYR (75 µM) > MAC (150 µM). Inhibition of gyrase activity was most pronounced for AOH and AME (initial inhibitory concentration 10 µM) followed by ATX II (25 µM) > ATX I = ALP = STTX III (50 µM). In contrast, none of the investigated Fusarium mycotoxins deoxynivalenol (DON), fumonisin B1, fusarin C and moniliformin, as well as the Alternaria metabolite tentoxin, had any impact on the activity of neither human nor bacterial topo II.

Activation of the Nrf2-ARE pathway by the Alternaria alternata mycotoxins altertoxin I and II.

The mycotoxins altertoxin I and II (ATX I and II) are secondary metabolites produced by Alternaria alternata fungi and may occur as food and feed contaminants, especially after long storage periods. Although the toxic potential of altertoxins has been previously investigated, little is known about the pathways that play a role in their intracellular metabolism. In order to identify potential targets of ATX I and ATX II, the two toxins were tested for interaction with the nuclear factor erythroid-derived 2-like 2/antioxidant response element (Nrf2/ARE) pathway in mammalian cells. This pathway can be activated by various stressors resulting in the expression of enzymes important for metabolism and detoxification. In the present study, only ATX II triggered a concentration-dependent increase in Nrf2-ARE-dependent luciferase expression. Consistently, confocal microscopy revealed an ATX II-induced increase in Nrf2 signal in HT29 intestinal cells. In agreement with these data, ATX II induced the transcription of γ-glutamate cysteine ligase, the key enzyme in catalyzing GSH synthesis of the cells and which is regulated by Nrf2. Further investigations demonstrated that ATX II induced a concentration-dependent depletion of the cellular GSH levels after short incubation time (3 h) and an increase after longer incubation time (24 h). In conclusion, it was demonstrated that ATX II can interact at several levels of the Nrf2-ARE pathway in mammalian cells and that ATX I does not share the same mechanism of action.

A Hydroalcoholic Extract from Paullinia pinnata L. Roots Exerts Anthelmintic Activity against Free-Living and Parasitic Nematodes.

Paullinia pinnata is a medicinal plant traditionally used in West Africa against a wide range of diseases including soil-transmitted helminthiases. In this study, a hydroethanolic root extract was investigated for its phytochemical composition and in vitro activity against the free-living nematode Caenorhabditis elegans as well as the larval stages of the parasitic helminths Ancylostoma caninum, Haemonchus contortus, Toxocara cati, and Trichuris vulpis.LC-MS analysis of the ethanol-water (1 : 1) extract revealed epicatechin and different A-type linked oligomeric and polymeric procyanidins as the predominant compounds.Within an in vitro mortality assay, the extract showed a lethal activity against T. cati (LC50 of 112 µg/mL), T. vulpis (LC50 of 17 µg/mL), and C. elegans (LC50 2.5 of mg/mL), but not against A. caninum. Additionally, effects on egg hatching and larval migration of H. contortus were investigated, but no inhibitory activity was observed.Overall, these findings rationalize the traditional use of the root extract from P. pinnata as an anthelmintic remedy and provide insight into the phytochemical composition of the extract.

Activation and modulation of recombinantly expressed serotonin receptor type 3A by terpenes and pungent substances.

Serotonin receptor type 3 (5-HT3 receptor) is a ligand-gated ion channel that is expressed in the central nervous system (CNS) as well as in the peripheral nervous system (PNS). The receptor plays an important role in regulating peristalsis of the gastrointestinal tract and in functions such as emesis, cognition and anxiety. Therefore, a variety of pharmacologically active substances target the 5-HT3 receptor to treat chemotherapy-induced nausea and vomiting. The 5-HT3 receptors are activated, antagonized, or modulated by a wide range of chemically different substances, such as 2-methyl-serotonin, phenylbiguanide, setrones, or cannabinoids. Whereas the action of all of these substances is well described, less is known about the effect of terpenoids or fragrances on 5-HT3A receptors. In this study, we screened a large number of natural odorous and pungent substances for their pharmacological action on recombinantly expressed human 5-HT3A receptors. The receptors were functionally expressed in Xenopus oocytes and characterized by electrophysiological recordings using the two-electrode voltage-clamp technique. A screening of two odorous mixes containing a total of 200 substances revealed that the monoterpenes, thymol and carvacrol, act as both weak partial agonists and positive modulators on the 5-HT3A receptor. In contrast, the most effective blockers were the terpenes, citronellol and geraniol, as well as the pungent substances gingerol, capsaicin and polygodial. In our study, we identified new modulators of 5-HT3A receptors out of the classes of monoterpenes and vanilloid substances that frequently occur in various plants.

Chemodiversity of cereulide, the emetic toxin of Bacillus cereus.

Food-borne intoxications are increasingly caused by the dodecadepsipeptide cereulide, the emetic toxin produced by Bacillus cereus. As such intoxications pose a health risk to humans, a more detailed understanding on the chemodiversity of this toxin is mandatory for the reliable risk assessment of B. cereus toxins in foods. Mass spectrometric screening now shows a series of at least 18 cereulide variants, among which the previously unknown isocereulides A-G were determined for the first time by means of UPLC-TOF MS and ion-trap MS(n) sequencing, (13)C-labeling experiments, and post-hydrolytic dipeptide and enantioselective amino acid analysis. The data demonstrate a high microheterogeneity in cereulide and show evidence for a relaxed proof reading function of the non-ribosomal cereulide peptide synthetase complex giving rise to an enhanced cereulide chemodiversity. Most intriguingly, the isocereulides were found to differ widely in their cell toxicity correlating with their ionophoric properties (e.g., purified isocereulide A showed about 8-fold higher cytotoxicity than purified cereulide in the HEp-2 assay and induced an immediate breakdown of bilayer membranes). These findings provide a substantial contribution to the knowledge-based risk assessment of B. cereus toxins in foods, representing a still unsolved challenge in the field of food intoxications.

Antioxidative compounds from Garcinia buchananii stem bark.

An aqueous ethanolic extract of the stem bark of Garcinia buchananii showed strong antioxidative activity using H2O2 scavenging, oxygen radical absorbance capacity (ORAC), and Trolox equivalent antioxidant capacity (TEAC) assays. Activity-guided fractionation afforded three new compounds, isomanniflavanone (1), an ent-eriodictyol-(3α→6)-dihydroquercetin-linked biflavanone, 1,5-dimethoxyajacareubin (2), and the depsidone garcinisidone-G (3), and six known compounds, (2″R,3″R)-preussianon, euxanthone, 2-isoprenyl-1,3,5,6-tetrahydroxyxanthone, jacareubin, isogarcinol, and garcinol. All compounds were described for the first time in Garcinia buchananii. The absolute configurations were determined by a combination of NMR, ECD spectroscopy, and polarimetry. These natural products showed high in vitro antioxidative power, especially isomanniflavanone, with an EC50 value of 8.5 µM (H2O2 scavenging), 3.50/4.95 mmol TE/mmol (H/L-TEAC), and 7.54/14.56 mmol TE/mmol (H/L-ORAC).

A new NMR approach for structure determination of thermally unstable biflavanones and application to phytochemicals from Garcinia buchananii.

Previous activity-guided phytochemical studies on Garcinia buchananii stem bark, which is traditionally used in Africa to treat various gastrointestinal and metabolic illnesses, revealed xanthones, polyisoprenylated benzophenones, flavanone-C-glycosides, biflavonoids, and/or biflavanones as bioactive key molecules. Unequivocal structure elucidation of biflavonoids and biflavanones by means of NMR spectroscopy is often complicated by the hindered rotation of the monomers around the C-C axis (atropisomerism), resulting in a high spectral complexity. In order to facilitate an unrestricted rotation, NMR spectra are usually recorded at elevated temperatures, commonly over 80 °C, which effects in a single set of resonance signals. However, under these conditions, one of the target compounds of this investigation, (2R,3S,2″R,3″R)-manniflavanone (1), undergoes degradation. Therefore, we demonstrated in the present study that the 1,1-ADEQUATE could be successfully used as a powerful alternative approach to confirm the C-C connectivities in 1, avoiding detrimental conditions. However, a moderate increase in temperature up to 50 °C was sufficient to deliver sharp signals in the proton NMR experiment of (2R,3S,2″R,3″R)-isomanniflavanone (2) and (2″R,3″R)-preussianone (3). In addition, two new compounds could be isolated, namely (2R,3S,2″R,3″R)-GB-2 7″-O-ß-D-glucopyranoside (4) and (2R,3S,2″R,3″R)-manniflavanone-7″-O-ß-D-glucopyranoside (5), and whose structures were elucidated by spectroscopic analysis including 1D and 2D NMR and mass spectrometry methods. The absolute configurations were determined by a combination of NMR and electronic circular dichroism (ECD) spectroscopy. The aforementioned compounds exhibited high anti-oxidative capacity in the H2O2 scavenging, hydrophilic Trolox equivalent antioxidant capacity (H-TEAC) and hydrophilic oxygen radical absorbance capacity (H-ORAC) assays.

A poisonous cocktail: interplay of cereulide toxin and its structural isomers in emetic Bacillus cereus.

Food intoxications evoked by emetic Bacillus cereus strains constitute a serious threat to public health, leading to emesis and severe organ failure. The emetic peptide toxin cereulide, assembled by the non-ribosomal peptide synthetase CesNRPS, cannot be eradicated from contaminated food by usual hygienic measures due to its molecular size and structural stability. Next to cereulide, diverse chemical variants have been described recently that are produced concurrently with cereulide by CesNRPS. However, the contribution of these isocereulides to the actual toxicity of emetic B. cereus, which produces a cocktail of these toxins in a certain ratio, is still elusive. Since cereulide isoforms have already been detected in food remnants from foodborne outbreaks, we aimed to gain insights into the composition of isocereulides and their impact on the overall toxicity of emetic B. cereus. The amounts and ratios of cereulide and isocereulides were determined in B. cereus grown under standard laboratory conditions and in a contaminated sample of fried rice balls responsible for one of the most severe food outbreaks caused by emetic B. cereus in recent years. The ratios of variants were determined as robust, produced either under laboratory or natural, food-poisoning conditions. Examination of their actual toxicity in human epithelial HEp2-cells revealed that isocereulides A-N, although accounting for only 10% of the total cereulide toxins, were responsible for about 40% of the total cytotoxicity. An this despite the fact that some of the isocereulides were less cytotoxic than cereulide when tested individually for cytotoxicity. To estimate the additive, synergistic or antagonistic effects of the single variants, each cereulide variant was mixed with cereulide in a 1:9 and 1:1 binary blend, respectively, and tested on human cells. The results showed additive and synergistic impacts of single variants, highlighting the importance of including not only cereulide but also the isocereulides in routine food and clinical diagnostics to achieve a realistic toxicity evaluation of emetic B. cereus in contaminated food as well as in patient samples linked to foodborne outbreaks. Since the individual isoforms confer different cell toxicity both alone and in association with cereulide, further investigations are needed to fully understand their cocktail effect.

Human Sensory, Taste Receptor, and Quantitation Studies on Kaempferol Glycosides Derived from Rapeseed/Canola Protein Isolates.

Beyond the key bitter compound kaempferol 3-O-(2‴-O-sinapoyl-ß-d-sophoroside) previously described in the literature (1), eight further bitter and astringent-tasting kaempferol glucosides (2-9) have been identified in rapeseed protein isolates (Brassica napus L.). The bitterness and astringency of these taste-active substances have been described with taste threshold concentrations ranging from 3.3 to 531.7 and 0.3 to 66.4 µmol/L, respectively, as determined by human sensory experiments. In this study, the impact of 1 and kaempferol 3-O-ß-d-glucopyranoside (8) on TAS2R-linked proton secretion by HGT-1 cells was analyzed by quantification of the intracellular proton index. mRNA levels of bitter receptors TAS2R3, 4, 5, 13, 30, 31, 39, 40, 43, 45, 46, 50 and TAS2R8 were increased after treatment with compounds 1 and 8. Using quantitative UHPLC-MS/MSMRM measurements, the concentrations of 1-9 were determined in rapeseed/canola seeds and their corresponding protein isolates. Depending on the sample material, compounds 1, 3, and 5-9 exceeded dose over threshold (DoT) factors above one for both bitterness and astringency in selected protein isolates. In addition, an increase in the key bitter compound 1 during industrial protein production (apart from enrichment) was observed, allowing the identification of the potential precursor of 1 to be kaempferol 3-O-(2‴-O-sinapoyl-ß-d-sophoroside)-7-O-ß-d-glucopyranoside (3). These results may contribute to the production of less bitter and astringent rapeseed protein isolates through the optimization of breeding and postharvest downstream processing.

Comparative direct infusion ion mobility mass spectrometry profiling of Thermus thermophilus wild-type and mutant ∆cruC carotenoid extracts.

The major carotenoid species isolated from the thermophilic bacterium Thermus thermophilus HB27 have been identified as zeaxanthin-glucoside-fatty acid esters (thermozeaxanthins and thermobiszeaxanthins). Most of the genes of the proposed T. thermophilus carotenoid pathway could be found in the genome, but there is less clarity about the genes which encode the enzymes performing the final carotenoid glycosylation and acylation steps. To get a further insight into the biosynthesis of thermo(bis)zeaxanthins in T. thermophilus, we deleted the megaplasmid open reading frame TT_P0062 (termed cruC) by both exchanging it with a kanamycin resistance cassette (ΔcruC:kat) and by generating a markerless gene deletion strain (ΔcruC). A fast and efficient electrospray ionization-ion mobility-time-of-flight mass spectrometry method via direct infusion was developed to compare the carotenoid profiles of wild type and mutant T. thermophilus cell culture extracts. These comparisons revealed significant alterations in the carotenoid composition of the ΔcruC mutant, which was found to accumulate zeaxanthin. This is the first experimental evidence that the ORF encodes the glycosyltransferase enzyme necessary for the glycosylation of zeaxanthin in the final modification steps of the thermozeaxanthin biosynthesis in T. thermophilus HB27. Also, the proposed method for direct determination of carotenoid amounts and species in crude acetone extracts represents an improvement over existing methods in terms of speed and sensitivity and may be applicable in high-throughput analyses of other terpenoids as well as other important bacterial metabolites like fatty acids and their derivatives.

Mass spectrometric profiling of Bacillus cereus strains and quantitation of the emetic toxin cereulide by means of stable isotope dilution analysis and HEp-2 bioassay.

A fast and robust high-throughput ultra-performance liquid chromatography/time-of-flight mass spectrometry (UPLC-TOF MS) profiling method was developed and successfully applied to discriminate a total of 78 Bacillus cereus strains into no/low, medium and high producers of the emetic toxin cereulide. The data obtained by UPLC-TOF MS profiling were confirmed by absolute quantitation of cereulide in selected samples by means of high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) and stable isotope dilution assay (SIDA). Interestingly, the B. cereus strains isolated from four vomit samples and five faeces samples from patients showing symptoms of intoxication were among the group of medium or high producers. Comparison of HEp-2 bioassay data with those determined by means of mass spectrometry showed differences, most likely because the HEp-2 bioassay is based on the toxic action of cereulide towards mitochondria of eukaryotic cells rather than on a direct measurement of the toxin. In conclusion, the UPLC-electrospray ionization (ESI)-TOF MS and the HPLC-ESI-MS/MS-SIDA analyses seem to be promising tools for the robust high-throughput analysis of cereulide in B. cereus cultures, foods and other biological samples.

Primary and Secondary Metabolites in Lotus japonicus.

Lotus japonicus is a leguminous model plant used to gain insight into plant physiology, stress response, and especially symbiotic plant-microbe interactions, such as root nodule symbiosis or arbuscular mycorrhiza. Responses to changing environmental conditions, stress, microbes, or insect pests are generally accompanied by changes in primary and secondary metabolism to account for physiological needs or to produce defensive or signaling compounds. Here we provide an overview of the primary and secondary metabolites identified in L. japonicus to date. Identification of the metabolites is mainly based on mass spectral tags (MSTs) obtained by gas chromatography linked with tandem mass spectrometry (GC-MS/MS) or liquid chromatography-MS/MS (LC-MS/MS). These MSTs contain retention index and mass spectral information, which are compared to databases with MSTs of authentic standards. More than 600 metabolites are grouped into compound classes such as polyphenols, carbohydrates, organic acids and phosphates, lipids, amino acids, nitrogenous compounds, phytohormones, and additional defense compounds. Their physiological effects are briefly discussed, and the detection methods are explained. This review of the exisiting literature on L. japonicus metabolites provides a valuable basis for future metabolomics studies.

Garcinia buchananii stem bark extract and its bioactive constituents manniflavanone, GB-2 and buchananiflavanone attenuate intestinal inhibitory neuromuscular transmission.

Garcinia buchananii stem bark extract (GBB), commonly used for treating diarrhea in Africa, triggers ectopic aboral contractions, causing inhibition of propulsive motility in the colon ex vivo. To determine whether or not these effects were associated with decreased inhibitory neuromuscular transmission, the responsible constituent compounds, and mechanisms of action, we studied the effects of GBB and specific fractions and flavanones isolated from GBB on intestinal motility using pellet propulsion assays in guinea pig distal colons. In addition, microelectrode recordings were used to measure the effects on the inhibitory junction potentials (IJPs) in the porcine ileum and descending colon smooth muscle. Psychoactive Drug Screening Program secondary receptor functional assays were used to determine whether or not GBB and its constituent compounds act via purinergic (P2Y) and muscarinic receptors. GBB inhibited propulsive motility, but (2R,3S,2″R,3″R)-manniflavanone (MNF), (2R,3S,2″R,3″R)-GB-2 (GB-2) and (2R,3S,2″S)-buchananiflavanone (BNF), the main ingredients of GBB, did not affect motility. We discovered that, in the porcine descending colon, IJPs contained purinergic, nitrergic, and nonpurinergic nonnitrergic components. Furthermore, ileal IJPs were purely purinergic. GBB blocked all components of IJPs, while MNF and GB-2 inhibited purinergic IJPs only. BNF inhibited the purinergic and nonpurinergic components of IJPs. MRS2365, a Y1 (P2Y) agonist, did not evoke sustained membrane hyperpolarization in the presence of GBB. However, GBB, MNF, GB-2 and BNF did not affect P2Y or muscarinic receptors. In conclusion, inhibitory neuromuscular transmission in the porcine descending colon involves all components of IJPs. GBB decreases inhibitory neuromuscular transmission, likely by the actions of MNF, GB-2 and BNF. These effects do not involve P2Y or muscarinic receptors.