Structural Impact of Steroidal Glycoalkaloids: Barrier Integrity, Permeability, Metabolism, and Uptake in Intestinal Cells.

SCOPE: Potato tubers represent an essential food component all over the world and an important supplier of carbohydrates, fiber, and valuable proteins. However, besides their health promoting effects, potatoes contain α-solanine and α-chaconine, which are toxic steroidal glycoalkaloids (SGAs). Other solanaceous plants like eggplants and tomatoes produce SGAs as well, different in their chemical structure. This study aims to investigate toxic effects (cholinesterase inhibition, membrane, and barrier disruption), permeability, metabolism, and structure-activity relationships of SGAs. METHODS AND RESULTS: α-solanine, α-chaconine, α-solasonine, α-solamargine, α-tomatine, and their respective aglycones solanidine, solasodine, and tomatidine are analyzed using Ellman assay, cellular impedance spectroscopy, cell extraction, and Caco-2 intestinal model. Additionally, metabolism is analyzed by HPLC-MS techniques. The study observes dependencies of barrier disrupting potential and cellular uptake on the carbohydrate moiety of SGAs, while permeability and acetylcholinesterase (AChE) inhibition are dominated by the steroid backbone. SGAs show low permeabilities across Caco-2 monolayers in subtoxic concentrations. In contrast, their respective aglycones reveal higher permeabilities, but are extensively metabolized. CONCLUSION: Besides structure-activity relationships, this study provides new information on the overall effects of steroidal alkaloids on intestinal cells and closes a gap of knowledge for the metabolic pathway from oral uptake to final excretion.

Characterization of Oligomeric Proanthocyanidin-Enriched Fractions from Aronia melanocarpa (Michx.) Elliott via High-Resolution Mass Spectrometry and Investigations on Their Inhibitory Potential on Human Topoisomerases.

Aronia melanocarpa (MICHX.) ELLIOTT, which belongs to the Rosaceae family, has increasingly come into focus of research due to the high content of polyphenols. In addition to antioxidative properties, further health-promoting effects of these polyphenols are still of interest. Especially, the proanthocyanidins offer thereby huge opportunities due to their high structural heterogeneity. Therefore, the present study focuses on the topoisomerase inhibiting effects of oligomeric proanthocyanidins (PACs), which are potentially depended on their degree of polymerization. The investigated PACs isolated from Aronia berries were characterized by chromatographic techniques and liquid chromatography-high-resolution mass spectrometry. Four PAC enriched fractions were obtained from Aronia pomace containing 47 PACs with a degree of polymerization from three to six. Due to the low yield of hexamers, the potential inhibiting effects against human topoisomerase were investigated for the trimer to pentamer fractions. The relaxation and decatenation assays were performed to examine the inhibiting effect on topoisomerases under cell-free conditions. Moreover, rapid isolation of topoisomerase cleavage complexes in human colon carcinoma HT29 cells was performed to evaluate the effect on topoisomerases in a cell-based system. The fractions demonstrated inhibitory potential on topoisomerases I and II. In sum, an increasing effect strength depending on the degree of polymerization was shown.

Identification of Novel Iso-Esculeoside B from Tomato Fruits and LC-MS/MS-Based Food Screening for Major Dietary Steroidal Alkaloids Focused on Esculeosides.

Plants from the Solanaceae family are known to be sources of several nutritionally relevant steroidal glycoalkaloids (SGAs). With the aim of quantitatively investigating the occurrence of the main SGA from tomatoes, eggplants, and potatoes in various food samples and evaluating their relevance in the human diet, a rapid single-step extraction liquid chromatography-tandem mass spectrometry method was developed. Over the course of isolating several commercially unavailable SGAs from tomato products to use them as reference standards, a previously unknown derivative was detected, structurally characterized, and identified as a novel isomer of esculeoside B-1 and B-2. After validation of the method, 36 food items exclusively derived from Solanaceae plants were analyzed for their SGA contents and a specific occurrence of each alkaloid in tomato, eggplant, or potato products was revealed. This is the first study reporting quantitative data on the occurrence of esculeoside A, B-1, B-2, and iso-esculeoside B in tomato products obtained by using appropriate reference compounds rather than applying a semi-quantitative approach based on α-tomatine as a reference. Some of the analyzed tomato products contained the esculeosides in concentrations of >500 mg/kg, clearly indicating their relevance in the human diet and the need of investigating their potential bioactivities in the future.

Occurrence of the Ochratoxin A Degradation Product 2'R-Ochratoxin A in Coffee and Other Food: An Update.

Food raw materials can contain the mycotoxin ochratoxin A (OTA). Thermal processing of these materials may result in decreased OTA levels but also in the formation of the thermal isomerization product 2'R-ochratoxin A (2'R-OTA). So far, only 2'R-OTA levels reported from 15 coffee samples in 2008 are known, which is little when compared to the importance of coffee as a food and trading good. Herein, we present results from a set of model experiments studying the effect of temperatures between 120 °C and 270 °C on the isomerization of OTA to 2'R-OTA. It is shown that isomerization of OTA starts at temperatures as low as 120 °C. At 210 °C and above, the formation of 25% 2'R-OTA is observed in less than one minute. Furthermore, 51 coffee samples from France, Germany, and Guatemala were analyzed by HPLC-MS/MS for the presence of OTA and 2'R-OTA. OTA was quantified in 96% of the samples, while 2'R-OTA was quantifiable in 35% of the samples. The highest OTA and 2'R-OTA levels of 28.4 µg/kg and 3.9 µg/kg, respectively, were detected in coffee from Guatemala. The OTA:2'R-OTA ratio in the samples ranged between 2.5:1 and 10:1 and was on average 5.5:1. Besides coffee, 2'R-OTA was also for the first time detected in a bread sample and malt coffee powder.

Detection of Novel Cytotoxic Imidazole Alkaloids in Tomato Products by LC-MS/MS.

Imidazole alkaloids represent a rather small group of alkaloids and are assumed not to be of significance to the human food chain so far. In this study, novel imidazole alkaloids occurring in tomato products were synthesized and structurally characterized by nuclear-magnetic-resonance spectroscopy and high-resolution mass spectrometry. These alkaloids are amides of either histidinol or histamine and short-chain fatty acids and could be quantitated in all of the 28 analyzed tomato products using a newly developed and validated LC-MS/MS-based method. Levels ranged from approximately 5 µg/kg to almost 3 mg/kg in the analyzed tomato products, and N-caprylhistidinol and its putative isomer were shown to occur in the highest amounts. These imidazole alkaloids are thus regularly ingested in considerable amounts as part of the human diet. In the course of evaluating their effects on the viability of HT-29 cells, all compounds were shown to significantly reduce cell viability starting at concentrations of 100 µM.

Large-Scale Screening of Foods for Glucose-Derived ß-Carboline Alkaloids by Stable Isotope Dilution LC-MS/MS.

The occurrence of glucose-derived ß-carboline alkaloids tangutorid E (Tan E) and tangutorid F (Tan F) as well as their dehydroxy-derivatives (DH-Tan E/F) was investigated in a broad variety of foodstuffs by LC-MS/MS-based stable isotope dilution analysis (SIDA). For that purpose, the target compounds and their 13C6-stable isotope-labeled analogues were synthesized from l-tryptophan and (13C6-)d-glucose and used to develop a rapid LC-MS/MS-SIDA method. After validation for several food matrices, the method was applied to the analysis of these ß-carbolines in 80 food items. Quantitative amounts were detected in 46.3, 50.0, and 42.5% of the samples regarding Tan E, Tan F, and DH-Tan E/F, respectively, with corresponding ranges of 0.01-6.75, 0.01-5.07, and 0.01-0.75 mg/kg; the highest amounts were found in processed tomato products. A combination of the obtained occurrence data in foods with average food consumption data led to the calculation of rough estimates for the chronic daily intake of those alkaloids, yielding values of 0.44, 0.36, and 0.13 µg/kg body weight/day for Tan E, Tan F, and DH-Tan E/F, respectively. Evidently, the consumption of processed tomato-based products accounts for the majority of the total daily intake of the investigated ß-carbolines; the potential bioactivities of Tan E, Tan F, and DH-Tan E/F have yet to be investigated.

Human Study on the Kinetics of 2'R-Ochratoxin A in the Blood of Coffee Drinkers.

SCOPE: The aim of this study is to obtain a deeper knowledge of the kinetics of 2'R-ochratoxin A (2'R-OTA), the thermal degradation product of the mycotoxin ochratoxin A (OTA). To investigate the correlation between the amount of this compound in roasted coffee and human blood samples, a human study is performed. METHODS AND RESULTS: An 18-week human study is carried out. During the first eight weeks, all known 2'R-OTA-containing food sources are excluded from the diet and the reduction of 2'R-OTA in venous blood is analyzed. Afterwards, participants are allowed to consume coffee with known OTA and 2'R-OTA concentrations. On a biweekly scale, 2'R-OTA and OTA blood levels are determined. After eight weeks of fasting on 2'R-OTA-containing foods, the 2'R-OTA blood concentration decreased by about 10%. Based on this, a long biological half-life of over seven months is estimated. In the 24 h urine samples collected before and after the coffee fasting period, only traces of 2'R-OTA are detected. CONCLUSION: Results show that 2'R-OTA has a more than seven-fold higher biological half-life in human blood compared to OTA (approx. 35 days). The reason for the long persistence of 2'R-OTA in human blood is still unclear and further research is needed.

Influence of Environmental Factors on the Production of Penitrems A-F by Penicillium crustosum.

Filamentous fungi produce a multitude of secondary metabolites, some of them known as mycotoxins, which are toxic to vertebrates and other animal groups in low concentrations. Among them, penitrems, which belong to the group of indole-diterpene mycotoxins, are synthesized by Penicillium and Aspergillus genera and exhibit potent tremorgenic effects. This is the first complex study of the penitrems A-F production under the influence of different abiotic factors, e.g., media, incubation time, temperature, pH, light, water activity, and carbon and nitrogen source as well as oxidative and salt stress. For this purpose, penitrems A-F were isolated from Penicillium crustosum cultures and used as analytical standards. Among the carbon sources, glucose supplemented to the media at the concentration of 50 g/L, showed the strongest inducing effect on the biosynthesis of penitrems. Among nitrogen sources, glutamate was found to be the most favorable supplement, significantly increasing production of these secondary metabolites. CuSO4-promoted oxidative stress was also shown to remarkably stimulate biosynthesis of all penitrems. In contrast, the salt stress, caused by the elevated concentrations of NaCl, showed an inhibitory effect on the penitrem biosynthesis. Finally, cheese model medium elicited exceptionally high production of all members of the penitrems family. Obtained results give insides into the biosynthesis of toxicologically relevant penitrems A-F under different environmental factors and can be utilized to prevent food contamination.

Sound of silence: the beauvericin cluster in Fusarium fujikuroi is controlled by cluster-specific and global regulators mediated by H3K27 modification.

In this study, we compared the secondary metabolite profile of Fusarium fujikuroi and the histone deacetylase mutant ΔHDA1. We identified a novel peak in ΔHDA1, which was identified as beauvericin (BEA). Going in line with a 1000-fold increased BEA production, the respective non-ribosomal peptide synthetase (NRPS)-encoding gene (BEA1), as well as two adjacent genes (BEA2-BEA3), were significantly up-regulated in ΔHDA1 compared to the wild type. A special role was revealed for the ABC transporter Bea3: deletion of the encoding gene resulted in significant up-regulation of BEA1 and BEA2 and drastically elevated product yields. Furthermore, mutation of a conserved sequence motif in the promoter of BEA1 released BEA repression and resulted in elevated product levels. Candidate transcription factors (TFs) that could bind to this motif are the cluster-specific TF Bea4 as well as a homolog of the global mammalian Kruppel-like TF Yin Yang 1 (Yy1), both acting as repressors of BEA biosynthesis. In addition to Hda1, BEA biosynthesis is repressed by the activity of the H3K27 methyltransferase Kmt6. Consistently, Western blot analyses revealed a genome-wide enrichment of H3K27 acetylation (H3K27ac) in the ΔHDA1 and KMT6 knock-down mutants. Subsequent chromatin immunoprecipitation (ChIP) experiments showed elevated H3K27ac modification levels at the BEA cluster.

New High-Performance Liquid Chromatography Coupled Mass Spectrometry Method for the Detection of Lobster and Shrimp Allergens in Food Samples via Multiple Reaction Monitoring and Multiple Reaction Monitoring Cubed.

Crustacean shellfish allergy ranks among the most frequent and severe food allergies for adults, demanding rugged and sensitive analytical routine methods. The objective of this study was therefore to develop a mass spectrometric approach for the detection of contamination with shrimp and lobster, two economically important types of crustaceans, in complex food matrices. Following a biomarker approach, we identified proteotypic peptides and developed a multiple reaction monitoring (MRM) method allowing for the identification and differentiation of shrimp and lobster in the food matrix at concentrations down to 0.1%. To further enhance sensitivity, we employed the MRM-cubed (MRM(3)) mode, which allowed us to detect crustaceans down to concentrations of 25 µg/g (crustacean/food, 0.0025%). We hereby present the first mass spectrometric method for the detection of shrimp and lobster in food matrices.

Baicalein modulates stress-resistance and life span in C. elegans via SKN-1 but not DAF-16.

The flavonoid baicalein has been demonstrated to be an activator of the transcription factor Nrf2 in mammalian cell lines. We show that it further modulates the Nrf2 homolog SKN-1 in Caenorhabditis elegans and by this pathway mediates beneficial effects in the nematode: baicalein enhances the resistance of C. elegans against lethal thermal and sodium arsenite stress and dose-dependently prolongs the life span of the nematode. Using RNA interference against SKN-1 we were able to show that the induction of longevity and the enhanced stress-resistance were dependent on this transcription factor. DAF-16 (homolog to mammalian FOXO) is another pivotal aging-related transcription factor in the nematode. We demonstrate that DAF-16 does not participate in the beneficial effects of baicalein: since baicalein causes no increase in the nuclear translocation of DAF-16 (DAF-16::GFP expressing strain, incubation time: 1h) and it still induces longevity even in a DAF-16 loss-of-function strain, we conclude, that baicalein increases stress-resistance and life span in C. elegans via SKN-1 but not DAF-16.

Modulation of the Nrf2 signalling pathway in Hct116 colon carcinoma cells by baicalein and its methylated derivative negletein.

CONTEXT: Baicalein is a major compound in extracts derived from Scutellaria baicalensis Georgi (Lamiaceae) which are used in the Traditional Chinese Medicine for the treatment of inflammatory and gastrointestinal diseases. This flavonoid is an activator of the Nrf2 signalling pathway but the molecular mechanism is not clearly established. OBJECTIVE: We investigated the molecular mode of baicalein-mediated Nrf2-activation in Hct116 cells by the analysis of proteasomal activity, radical-scavenging activity and the comparison with baicalein derivatives. MATERIALS AND METHODS: The radical-scavenging activity (TEAC, DCF) up to 25 µM, cytotoxicity (MTT assay, 48 h) up to 100 µM, proteasomal activity and the Nrf2-activation (luciferase assay, ubiquitinylation, western blot, Ser40-phosphorylation; incubation for 1 or 4 h) by concentrations up to 40 or 50 µM of the compounds were analysed in Hct116 human colon carcinoma cells. RESULTS: No change in the ubiquitinylation of Nrf2, proteasomal activity and transcription of the NRF2 gene were detectable. Baicalein decreased the phosphorylation of Nrf2 (IC50-value approximately 20 µM) suggesting an inhibitory effect of the flavonoid on protein kinases. Since the activation of the Nrf2 pathway by baicalein might be also due to redox-activity of the compound, we investigated the effects of methylated baicalein derivatives oroxylin A, negeletein and baicaleintrimethylether. Oroxylin A and negletein showed a comparable redox-active potential, but only negletein (50 µM, 4 h) was able to activate Nrf2. CONCLUSION: This result confirms the hypothesis that baicalein, a component of extracts derived from Baical Skullcap, causes an activation of Nrf2 independent of a modulation of the cellular redox potential.

Biomonitoring using dried blood spots: detection of ochratoxin A and its degradation product 2'R-ochratoxin A in blood from coffee drinkers.

SCOPE: In this study, human exposure to the mycotoxin ochratoxin A (OTA) and its thermal degradation product 2'R-ochratoxin A (2'R-OTA, previously named as 14R-Ochratoxin A [22]) through coffee consumption was assessed. LC-MS/MS and the dried blood spot (DBS) technique were used for the analysis of blood samples from coffee and noncoffee drinkers (n = 50), and food frequency questionnaires were used to document coffee consumption. METHODS AND RESULTS: For the detection of OTA and 2'R-OTA in blood, a new sensitive and efficient sample preparation method based on DBS was established and validated. Using this technique 2'R-OTA was for the first time detected in biological samples. Comparison between coffee drinkers and noncoffee drinkers showed for the first time that 2'R-OTA was only present in blood from the first group while OTA could be found in both groups in a mean concentration of 0.21 µg/L. 2'R-OTA mean concentration was 0.11 µg/L with a maximum concentration of 0.414 µg/L. Thus, in average 2'R-OTA was approx. half the concentration of OTA but in some cases even exceeded OTA levels. No correlation between the amounts of coffee consumption and OTA or 2'R-OTA levels was observed. CONCLUSION: The results of this study revealed for the first time a high exposure of coffee consumers to 2'R-OTA, a compound formed from OTA during coffee roasting. Since little information is available regarding toxicity and possible carcinogenicity of this compound, further OTA monitoring in blood including 2'R-OTA is advisable.

Caffeic acid phenethylester increases stress resistance and enhances lifespan in Caenorhabditis elegans by modulation of the insulin-like DAF-16 signalling pathway.

UNLABELLED: CAPE is an active constituent of propolis which is widely used in traditional medicine. This hydroxycinnamic acid derivate is a known activator of the redox-active Nrf2 signalling pathway in mammalian cells. We used C. elegans to investigate the effects of this compound on accumulation of reactive oxygen species and the modulation of the pivotal redox-active pathways SKN-1 and DAF-16 (homologues of Nrf2 and FoxO, respectively) in this model organism; these results were compared to the effects in Hct116 human colon carcinoma cells. CAPE exerts a strong antioxidative effect in C. elegans: The increase of reactive oxygen species induced by thermal stress was diminished by about 50%. CAPE caused a nuclear translocation of DAF-16, but not SKN-1. CAPE increased stress resistance of the nematode against thermal stress and finally a prolongation of the median and maximum lifespan by 9 and 17%, respectively. This increase in stress resistance and lifespan was dependent on DAF-16 as shown in experiments using a DAF-16 loss of function mutant strain. Life prolongation was retained under SKN-1 RNAi conditions showing that the effect is SKN-1 independent. The results of CAPE obtained in C. elegans differed from the results obtained in Hct116 colon carcinoma cells: CAPE also caused strong antioxidative effects in the mammalian cells, but no activation of the FoxO4 signalling pathway was detectable. Instead, an activation of the Nrf2 signalling pathway was shown by luciferase assay and western blots. CONCLUSION: CAPE activates the insulin-like DAF-16, but not the SKN-1 signalling pathway in C. elegans and therefore enhances the stress resistance and lifespan of this organism. Since modulation of the DAF-16 pathway was found to be a pivotal effect of CAPE in C. elegans, this has to be taken into account for the investigation of the molecular mechanisms of the traditional use of propolis.

In vitro metabolism studies of erythraline, the major spiroalkaloid from Erythrina verna.

BACKGROUND: Erythrina verna, popularly known as "mulungu", is a Brazilian medicinal plant used to treat anxiety. Erythrina alkaloids have been described in several species of Erythrina, which have biological and therapeutic properties well known that include anxiolytic and sedative effects. METHODS: In this work, in vitro metabolism of erythraline (1), the major spirocyclic alkaloid of Erythrina verna, was studied in the pig cecum model and by biomimetic phase I reactions. The biomimetic reactions were performed with Jacobsen catalyst to produce oxidative metabolites and one metabolite was isolated and evaluated against cancer cells, as HL-60 (promyelocytic leukemia), SF-295 (Glioblastoma) and OVCAR-8 (ovarian carcinoma). RESULTS: Erythraline exhibited no metabolization by the pig microbiota and a main putative metabolite was formed in a biomimetic model using Jacobsen catalyst. This metabolite was isolated and identified as 8-oxo-erythraline (2). Finally, erythraline and the putative metabolite were tested in MTT model and both compounds showed no important cytotoxic activity against tumor cells. CONCLUSIONS: The alkaloid erythraline was not metabolized by intestinal microbiota, but it was possible to identify its oxidative metabolite from biomimetic reactions. So these data are interesting and stimulate other studies involving this alkaloid, since it is present in phytomedicine products and there are not reported data about the metabolism of erythrina alkaloids.

Sphingomyelin and phosphatidylcholine contrarily affect the induction of apoptosis in intestinal epithelial cells.

SCOPE: The major alimentary sources for the plasma membrane lipid sphingomyelin (SM) are dairy products, eggs, and meat. We recently reported that the SM metabolite ceramide induces cathepsin D mediated apoptosis in murine intestinal epithelial cells (IECs) and increases inflammation in acute colitis. We investigated the impact of SM and phosphatidylcholine on apoptosis in human IECs and point out BH3-interacting death agonist (BID) as link between cathepsin D and apoptosis. METHODS AND RESULTS: HT-29 and isolated human IECs were stimulated with SM or phosphatidylcholine. SM treatment resulted in increased apoptosis. Phosphatidylcholine showed contrary effects. Western revealed higher amounts of cathepsin D and BID activation upon lipid stimulation. Western blotting revealed BID activation through SM in both an induced and a spontaneous mouse model of colitis. CONCLUSION: Dietary phospholipids may induce or abolish apoptosis in IECs and seem to play a role in the pathogenesis of inflammatory bowel diseases. This nutritional factor might be considered when evaluating the pathogenesis of inflammatory bowel diseases. Effects of SMase- and SM treatment on inflammation in dextran sulfate sodium induced animal models of colitis and in vitro experiments are discussed as controversial. Variable sources of SM, feeding techniques, and mouse strains might play a role.

Distribution and quantification of flavan-3-ols and procyanidins with low degree of polymerization in nuts, cereals, and legumes.

The monomeric flavan-3-ols catechin and epicatechin as well as procyanidins are of great interest due to their potential beneficial health effects observed in epidemiological studies. However, the occurrence and concentration of these compounds is not well-known due to the fact that reference compounds are not commercially available. In this study we determined the pattern and concentration of catechin, epicatechin, and different dimeric and trimeric procyanidins in 38 food samples (nuts, cereals, legumes) using a reversed phase high-performance liquid chromatography electrospray ionization tandem mass spectrometry (RP-HPLC-ESI-MS/MS) approach based on isolated authentic reference compounds. Of the analyzed food samples 21 were found to contain dimeric and trimeric procyanidins and their monomeric building units catechin and epicatechin. Mainly the monitored nut samples contained the analyzed procyanidins as well as catechin and epicatechin whereas only 3 cereals were identified as sources of the analyzed compounds. The concentration ranged from 148 µg/100 g in macadamia nut to 55 mg/100 g in pinto bean. Catechin and procyanidin B3 were found to be the most abundant analytes. The only A-type procyanidin that could be identified was procyanidin A2, which was found in peanut. The achieved data could be used for authenticity control and furthermore in combination with dietary studies to calculate the daily intake of monomeric flavan-3-ols and procyanidins. To our knowledge this is the first detailed study quantifying monomeric flavan-3-ols and dimeric and trimeric procyanidins in various nuts, cereals, and legumes.