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Selected technological characteristics and bioactive compounds of juice pressed directly from the mash of whole Opuntia dillenii cactus fruits have been investigated. The impact of pulsed electric fields (PEF) for a non-thermal disintegration on the important juice characteristics has been evaluated in comparison to microwave heating and use of pectinases. Results showed that the cactus juice exhibited desirable technological characteristics. Besides, it also contained a high amount of phenolic compounds being the major contributors to the overall antioxidant activity of juice. HPLC-DAD/ESI-MS n measurements in the fruits’ peel and pulp showed that isorhamnetin 3- O -rutinoside was determined as the single flavonol found only in the fruit’s peel. Treating fruit mash with a moderate electric field strength increased juice yield and improved juice characteristics. Promisingly, the highest release of isorhamnetin 3- O -rutinoside from fruit’s peel into juice was maximally achieved by PEF.

Toxin-producing cyanobacterial species are increasingly being found in freshwater systems. However, literature on the impact of many cyanobacterial toxins on plants is scarce. Cylindrospermosin (CYN), a secondary metabolite of cyanobacteria such as Cylindrospermopsis and Aphanizomenon species, is a potent hepatotoxin and protein synthesis inhibitor. Worryingly, CYN is increasingly found in surface and drinking water worldwide causing human and animal intoxications. Further, exposure of crop plants to CYN by irrigation with contaminated water has already been shown. Therefore, in this study, horticulturally important and highly consumed Brassica species were investigated to determine the level of CYN in the leaves after exposure of the roots to the toxin. Treatment of Brassica oleracea var. sabellica , Brassica juncea , and Sinapis alba under varying experimental conditions showed significant CYN uptake, with CYN levels ranging from 10% to 21% in the leaves compared to the CYN concentration applied to the roots (18–35 μg/l). In seedlings, CYN concentrations of up to 49 μg/g fresh weight were observed. Thus, crop plants irrigated with CYN-containing water may represent a significant source of this toxin within the food chain.

a b s t r a c t The winter crop kale has a complex profile of different glycosylated and acylated flavonol glycosides which may be affected by global warming. To the best of our knowledge, compound-climate relation- ships for flavonol aglycones and flavonol glycosides were established for the first time. The investigated 10 major flavonol glycosides responded structure-dependent in the investigated temperature range between 0 and 12 °C and the photosynthetic active radiation range between 4 and 20 mol m y 2 d y 1 , e.g. the decrease in temperature led to an increase in sinapic acid monoacylated and diacylated quercetin gly- cosides, while the sinapic acid monoacylated kaempferol glycosides showed a maximum at 4.5 °C. Fur- thermore, the hydroxycinnamic acid residues and the different number of glucose moieties in the 7- O position affected the response of kaempferol triglucosides. Consequently, global warming would result in lower concentrations of antioxidant-relevant quercetin glycosides in winter crops, suggesting a pro- duction at e.g. higher altitudes due to lower temperature.

The popularity of white asparagus (Asparagus officinalis L.) spears is mainly due to its unique flavour and its high concentration of health promoting compounds. Physical elicitors that activate plant defence systems are known to cause an increase of antioxidant compounds in plant tissue, such as phenolic compounds, and their corresponding enzymes (e.g. phenylalanine ammonia- lyase, [PAL, EC 4.3.1.5], peroxidase [POD, EC 1.11.1.7]). The objective of this work was to evaluate the impact of UV-B on PAL and POD kinetics, as well as to characterize the resulting changes in flavonoid composition in the different morphological sections (apical and basal) of harvested asparagus spears. For this purpose, spears of the cultivar ‘Gijnlim’ were irradiated with either 0.54 kJ m− 2 or 1.08 kJ m− 2 of UV-B. In order to follow the temporal dynamics of the UV-B treatments, analysis were performed after 2 h and 22 h of adaptation. Results revealed changes in the polyphenol composition, and in the activity of related enzymes (PAL, POD), in the different morphological parts of asparagus spears as mediated by UV-B dosage at the different adaptation times. The flavonol quercetin-4′-O-monoglucoside showed the most pronounced UV-B response because its concentration increased with increasing UV-B dosage. Furthermore, POD activity of both segments tended to increase with higher UV-B dosage. In contrast, PAL activity in the base segment tended to decrease with higher UV-B irradiation, while PAL activity in tips remained unaffected. However, PAL and POD activities were mainly influenced by the adaptation time. While PAL decreased with the duration of adaptation time, POD showed an opposite behaviour. The presented results revealed the complexity of the antioxidant defence system of asparagus, which is discussed in detail.

Glucosinolates, present in Brassica vegetables, are thought to contribute to human health prevention because of their enzymatically induced breakdown products, primarily isothiocyanates (ITCs). ITCs are reactive substances that readily react with nucleophilic (food) compounds. The reactivity of allyl-ITC and 4-(methylsulfinyl)butyl-ITC (sulforaphane) toward thiol and amino groups of cysteine and lysine derivatives was studied in buffered model systems as well as broccoli sprouts. The thiol group is the preferred reaction site, and it was demonstrated that even endogenously released sulforaphane is able to react very fast with cysteine in broccoli sprouts. Amino groups reacted slower and only under basic conditions. However, great differences in the reactivity between the different amino compounds were revealed. The aliphatic allylamine reacted very fast with allyl-ITC, forming N,N'-diallylthiourea, a compound identified as a main thermal degradation product of allyl-ITC.

Kale is a common Brassica vegetable rich in secondary plant metabolites especially flavonols, that comprise highly glycosylated and acylated quercetin and kaempferol derivatives. Several kale flavonols have been isolated, structurally identified and characterized by means of HPLC-DAD-ESI–MSn and NMR spectroscopy from kale for the first time. Their antioxidant activity given by a wide variety of glycosylation and acylation patterns was investigated using the Folin–Ciocalteu assay, the TEAC assay, and EPR spectroscopy. The purpose was to obtain distinct structure–antioxidant activity relationships of structures differing either in their aglycone structure, their number of glycoside substituent, and the kind of acylation. A high radical scavenging activity is mainly due to catechol like structure of e.g. quercetin derivatives. Noteworthy is the fact that flavonol derivatives esterified with methoxylated hydroxycinnamic acids possess higher antioxidant capacities than those conjugated with hydroxycinnamic acids with catechol structure. Remarkably, the influence of the acyl moiety seems to be less effective than that of the glycosylation pattern in 7-O position. In conclusion, the antioxidant behavior differs depending on the assay used and the above mentioned structural features, revealing the importance of these attributes to be responsible for a specific antioxidant capacity.

Glucosinolates are secondary plant metabolites occurring in Brassica vegetables. Food processing significantly reduces glucosinolate content, among other things due to thermal degradation. As there is only little information about thermal glucosinolate breakdown, the influence of the chemical structure as well as the influence of different pH to thermal degradation of individual glucosinolates was studied by analysing desulphoglucosinolates with HPLC-DAD. Thermal degradation was forced by heating broccoli sprouts at 130 °C in dry medium, whereas the influence of the pH was studied by cooking at 100 °C in aqueous medium . Within each group of glucosinolates differences in thermal degradation were revealed. Within the sulphur containing aliphatic glucosinolates the oxidative state of the sulphur atom as well as the side chain length influenced the reactivity. Among the indole glucosinolates great differences in stability were observed. A hydroxyl function in the side chain generally seems to destabilise glucosinolates. Glucosinolates were most stable towards thermal treatment in neutral and slightly acidic medium, whereas they degraded more rapidly in basic medium.

The objective of this study was to investigate the impact of mycorrhizal symbiosis on qualitative characteristics of onion (Allium cepa L.). For this reason, five onion cultivars with different scale color and three different strains of arbuscular mycorrhizal fungi (Diversispora versiformis, Rhizophagus intraradices, Funneliformis mosseae) were used. Red cultivars, mainly 'Red Azar-shahr', showed the highest content in vitamin C, flavonols, and antioxidant enzymes. Mycorrhizal inoculation increased total phenolic, pyruvic acid, and vitamin C of onion plants. Considerable increase was observed in quercetin-4'-O-monoglucoside and isorhamnetin-4'-O-monoglucoside content in plants inoculated with Diversispora versiformis, but quercetin-3,4'-O-diglucoside was not significantly influenced. Analyses for phenylalanine ammonia-lyase (PAL) and antioxiodant enzyme activities such as polyphenol oxidase (PPO), catalase (CAT), and peroxidase (POD) revealed that all except PPO were enhanced by mycorrhizal inoculation. Overall, these findings suggested that mycorrhizal inoculation influenced biosynthesis of flavonol glucosides and antioxidant enzymes by increasing nutrient uptake or by induction of the plant defense system.

The antimicrobial effectiveness of non-thermal plasma is scientifically accepted. Applications can be found in medicinal sterilisation processes. However, the effects of plasma treatments on plant food with regard to nutritional value are not yet sufficiently investigated. To study the interactions of plasma immanent reactive species with secondary plant metabolites, lamb’s lettuce (Valerianella locusta) was exposed to an atmospheric pressure plasma jet. Changes in the phenolic profile were compared to experiments with pure substances to determine the influence of the food matrix. Whereas pure flavonoids showed a strong time-dependent decrease, not originating from photo- or thermodesorption processes, the content of diosmetin in lamb’s lettuce significantly increased. Regarding phenolic acids, plasma exposure led to a strong reduction in lamb’s lettuce, while the concentration of pure monophenols changed little within the same period of time. Treatments with plasma-similar temperatures and UV radiation suggest that due to plasma-reactive species concomitant disintegration of cell membranes and oxidation of released cellular compounds are taking place. Time-dependent changes of the leaf surface morphology due to plasma-induced erosion have been observed by scanning electron microscopy.

The intestinal absorption of tocotrienols (TCT) in dogs is, to our knowledge, so far unknown. Adult Beagle dogs (n 8) were administered a single oral dosage of a TCT-rich fraction (TRF; 40 mg/kg body weight) containing 32 % a-TCT, 2 % b-TCT, 27 % g-TCT, 14 % d-TCT and 25 % a-tocopherol (a-TCP). Blood was sampled at baseline (fasted), 1, 2, 3, 4, 5, 6, 8 and 12 h after supplementation. Plasma and chylomicron concentrations of TCT and a-TCP were measured at each time point. Plasma TAG were measured enzymatically, and plasma antioxidant capacity was assessed by the Trolox equivalent antioxidant capacity assay. In fasted dogs, levels of TCT were 0·07 ( SD 0·03) mmol/l. Following the administration of the TRF, total plasma TCT peaked at 2 h (7·16 ( SD 3·88) mmol/l; P, 0·01) and remained above baseline levels (0·67 ( SD 0·44) mmol/l; P, 0·01) at 12 h. The TCT response in chylomicrons paralleled the increase in TCT in plasma with a maximum peak (3·49 ( SD 2·06) mmol/l; P, 0·01) at 2 h post-dosage. a-TCP was the major vitamin E detected in plasma and unaffected by TRF supplementation. The Trolox equivalent values increased from 2 h (776 ( SD 51·2) mmol/l) to a maximum at 12 h (1130 ( SD 7·72) mmol/l; P,0·01). The results show that TCT are detected in postprandial plasma of dogs. The increase in antioxidant capacity suggests a potential beneficial role of TCT supplementation in the prevention or treatment of several diseases in dogs., Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe, 571

Flavonols, betacyanins content and antioxidant activity of red-purple cactus Opuntia macrorhiza fruits, a promising cactus pear species, were evaluated in comparison to the well known cactus pear fruits of Opuntia ficus-indica. Flavonol profile was evaluated by HPLC-DAD prior to and after enzymatic hydrolysis (glycosides vs. aglycons). In addition betacyanins, responsible for the purple to red color of cactus pear fruits were also estimated and compared to Beta vulgaris ssp. (roots) and red O. ficus-indica fruits. In all cases, cactus fruit pulps showed no flavonols at all. While different derivatives of isorhamnetin glycosides were found in O. ficus-indica fruit's peel, isorhamnetin-3-O-rutinoside was the only compound in O. macrorhiza fruit. Correspondingly, antioxidant activity assays showed a high antioxidant activity of both, O. macrorhiza fruit's peel and pulp. With regard to betacyanins, O. macrorhiza fruit's peel and pulp provide a deep red-purple color, whose average impact is higher compared to red beet (B. vulgaris spp.) and about 8-fold higher than red fruits from O. ficus-indica. Supporting these results, estimation of color attributes (L*, a*, b*, C and H°) showed also a high color impact of both O. macrorhiza fruit's peel and pulp.

There are many reports on the potential consequences of UV-B radiation on plants, but there is a rather limited understanding of the effect on secondary plant metabolites, e.g. phenolic compounds and volatiles, at all. The popularity of highbush blueberries (Vaccinium corymbosum L.) is mainly due to its unique flavour and its high content of bioactive compounds, i.e. phenolic compounds. However, information on UV-B elicitor mediated changes on secondary plant metabolites on blueberries is scanty. In the present study, blueberry fruits were harvested and exposed to UV-B radiation with different dosage and adaptation times. With regard to volatile secondary metabolites, C6-aldehydes, terpenes and ketones, an increase of the relative peak area was observed after both UV-B treatments (0.075 and 0.15 Wh/m2 = low [L] and high [H] dosage, respectively). Furthermore, there was a strong influence of the adaptation time. Increasing relative peak areas were determined already after a short adaptation time (2 h) at both, low and high UV-B dosages, but after 24 h adaptation time relative peak areas decreased significantly. However, alcoholic compounds, as degradation products of aldehydes, showed opposite results. In contrast, the non-volatile phenolic compounds revealed a continuously increase with UV-B intensity.

The applicability of pectinases and cellulases as soft hydrolysing agents on flavonol glycosides was investigated for identification and quantification of flavonol aglycons in cactus pear fruit. Freeze-dried samples of cactus pear fruit’s peel (cactus pear peels) and onions were treated with commercial pectinase and cellulase preparations at 50 °C for different time periods (up to 16 h). Additionally isorhamnetin-3- O -rutinoside and quercetin-3,4′- O -diglucoside were used as model compounds. In parallel, samples of cactus pear peels and onions were treated by the traditional acidic hydrolysis using HCl. Following hydrolysis, flavonols were characterised using HPLC–DAD. Enzymatically, all model compounds and plant material were highly hydrolysable. Flavonol glycosides were completely hydrolysed after 16 h (cactus pear) and 4 h (onion), respectively. While the acidic hydrolysis caused degradation of the flavonols and produced protocatechuic acid as a degradation product, the enzymatic hydrolysis gave gentle effects and did not produce any protocatechuic acid at all.

Flavonoids have gained much attention because of their proposed positive effects for human health. Onions are a rich source of flavonoids, consisting mainly of the major flavonols quercetin-3,4′- O -diglucoside (QDG) and quercetin-4′- O -monoglucoside (QMG) in the bulb and the aglycone quercetin in the outer scales. In this study, distribution of these three flavonoids was determined in 16 onion cultivars ( Allium cepa ) using HPLC–DAD. Three different parts of the onion bulb, the inner layers, the middle layers and the outer scales were analysed. The analysis showed varietal differences in the selected onion cultivars. The cultivars with the highest total flavonoid content were the red skinned ‘Red Baron’ and the yellow skinned cultivars ‘Ailsa Craig’ and ‘Prilep’. The distribution of the total flavonoid content in the different parts of the onion bulb showed the following order: middle layers > outer scales > inner layers. In the inner layers QDG was the major flavonoid, while in the middle layers QDG and QMG were in equal amounts. In the outer scales quercetin was the major flavonoid prior to QMG.

The influence of a low-pressure oxygen glow discharge plasma on secondary metabolites of lamb’s lettuce was examined. After extraction and hydrolysis the compounds were analysed by means of RP-HPLC. Whereas pure compounds showed a time-dependent degradation (flavonoids) or remained unchanged (phenolic acids), the exposure of lamb’s lettuce to the oxygen plasma led to a significant increase of protocatechuic acid, luteolin, and disometin after 120 s treatment time, independent of the applied plasma driving voltage. Contact angle (CA) measurements show an increased wettability of adaxial leaf surfaces after plasma exposure. A successive degradation of epicuticular waxes and cutin of the plant’s epidermis has been indicated by means of FTIR (ATR) and scanning electron microscopy (SEM). These findings suggest that reactive species in the plasma play a crucial role for the observed changes upon exposure to non-thermal plasma.

Scope Different metabolic and excretion pathways of the benzyl glucosinolate breakdown products benzyl isothiocyanate and benzyl cyanide are investigated to obtain information about their multiple fate after ingestion. Detailed focus is on the so far underestimated transformation/excretion pathways-protein conjugation and exhalation. Methods and results Metabolites, protein conjugates, and non-conjugated isothiocyanates are determined in plasma, urine, and breath of seven volunteers after consuming freeze-dried nasturtium or bread enriched with nasturtium. Samples are collected up to 48 h at selected time points. The metabolites of the mercapturic acid pathway are detectable in plasma up to 24 h after consumption. Additionally, mercapturic acid is the main metabolite in urine, but non-conjugated benzyl isothiocyanate is detectable as well. Protein conjugates show high amounts in plasma even 48 h after consumption. In breath, benzyl isothiocyanate and benzyl cyanide are detectable up to 48 h after consumption. Conclusion Isothiocyanates are not only metabolized via the mercapturic acid pathway, but also form protein conjugates in blood and are exhaled. To balance intake and excretion, it is necessary to investigate all potential metabolites and excretion routes. This has important implications for the understanding of physiological and pharmacological effects of isothiocyanate-containing products.

Kale is a member of the Brassicaceae family and has a complex profile of flavonoid glycosides. Therefore, kale is a suitable matrix to discuss in a comprehensive study the different fragmentation patterns of flavonoid glycosides. The wide variety of glycosylation and acylation patterns determines the health-promoting effects of these glycosides. The aim of this study is to investigate the naturally occurring flavonoids in kale. A total of 71 flavonoid glycosides of quercetin, kaempferol and isorhamnetin were identified using a high-performance liquid chromatography diode-array detection/electrospray ionization multi-stage mass spectrometry (HPLC-DAD/ESI-MS(n)) method. Of these 71 flavonol glycosides, 27 were non-acylated, 30 were monoacylated and 14 were diacylated. Non-acylated flavonol glycosides were present as mono-, di-, tri- and tetraglycosides. This is the first time that the occurrence of four different fragmentation patterns of non-acylated flavonol triglycosides has been reported in one matrix simultaneously. In addition, 44 flavonol glycosides were acylated with p-coumaric, caffeic, ferulic, hydroxyferulic or sinapic acid. While monoacylated glycosides existed as di-, tri- and tetraglycosides, diacylated glycosides occurred as tetra- and pentaglycosides. To the best of our knowledge, 28 compounds in kale are reported here for the first time. These include three acylated isorhamnetin glycosides (isorhamnetin-3-O-sinapoyl-sophoroside-7-O-D-glucoside, isorhamnetin-3-O-feruloyl-sophoroside-7-O-diglucoside and isorhamnetin-3-O-disinapoyl-triglucoside-7-O-diglucoside) and seven non-acylated isorhamnetin glycosides.

The aim of this study was to determine the composition and concentration of flavonoid aglycones in kale, the dependence on genotype and their interaction with decreasing temperature and global radiation. Eight kale cultivars, comprising hybrid and traditional, old cultivars, were grown in a field experiment and harvested four times at 4-week intervals. The traditional, old cultivars in particular contained high concentrations of flavonoids. In all of the investigated cultivars, kaempferol was the main flavonoid aglycone, followed by quercetin and isorhamnetin, which was quantified in six of the eight cultivars. Furthermore, in six of the eight cultivars, the total concentration of flavonoids remained unchanged with decreasing temperature and global radiation. The quercetin concentration increased in five of these six cultivars, whereas the kaempferol concentration decreased. Interestingly, the quercetin-to-kaempferol ratio increased in all of the investigated cultivars, despite the fact that the radiation level decreased, suggesting that the impact of the decline in temperature could be responsible for this effect.

In the last decade, electrochemical oxidation coupled with mass spectrometry has been successfully used for the analysis of metabolic studies. The application focused in this study was to investigate the redox potential of different phenolic compounds such as the very prominent chlorogenic acid. Further, EC/ESI-MS was used as preparation technique for analyzing adduct formation between electrochemically oxidized phenolic compounds and food proteins, e.g., alpha-lactalbumin or peptides derived from a tryptic digestion. In the first step of this approach, two reactant solutions are combined and mixed: one contains the solution of the digested protein, and the other contains the phenolic compound of interest, which was, prior to the mixing process, electrochemically transformed to several oxidation products using a boron-doped diamond working electrode. As a result, a Michael-type addition led to covalent binding of the activated phenolic compounds to reactive protein/peptide side chains. In a follow-up approach, the reaction mix was further separated chromatographically and finally detected using ESI-HRMS. Compound-specific, electrochemical oxidation of phenolic acids was performed successfully, and various oxidation and reaction products with proteins/peptides were observed. Further optimization of the reaction (conditions) is required, as well as structural elucidation concerning the final adducts, which can be phenolic compound oligomers, but even more interestingly, quite complex mixtures of proteins and oxidation products.

1,4-Benzopyrone derivates are exposed to different cold gas discharges. Reactions are carried out using different feed gases (argon, oxygen) and excitation sources (radio frequency, microwave) at both atmospheric and low pressure. A structure-dependent degradation upon plasma-chemical reactions can be observed. From contact-angle measurements a strong surface oxidation is suggested. Independent of the source used plasma treatment leads to a significant increase in oxygen content of the samples through newly introduced carbonyland carboxyl-functions. This is in agreement to results showing that during thermal food processes oxidative species lead to the formation of characteristic low-molecular weight degradation products.

The influence of genotype and climatic factors, e.g. mean temperature and mean global radiation level, on the antioxidant activity of kale was investigated. Therefore, eight kale cultivars, hybrid and traditional, old cultivars, were grown in a field experiment and harvested at four different times. In addition to the investigation of the total phenolic content, the overall antioxidant activity was determined by TEAC assay and electron spin resonance spectrometry. A special aim was to characterize the contribution of single flavonoids to the overall antioxidant activity using an HPLC-online TEAC approach. The antioxidant activity and the total phenolic content were influenced by the genotype and the eco-physiological factors. The HPLC-online TEAC results showed that not all flavonol glycosides contribute to the overall antioxidant activity in the same manner. Taking the results of the structural analysis obtained by HPLC-ESI-MS(n) into account, distinct structure-antioxidant relationships have been observed.

The phenolic profile and quantitative composition of blueberries as well as the corresponding antioxidant activity of blueberries is well documented. Unfortunately, little is reported on the development of phenolic compounds and antioxidant activity during fruit maturation and ripening. In the present study, the total phenolic content and main phenolic compounds of four highbush blueberry cultivars (Vaccinium corymbosum L.) were analyzed at five stages of maturation and ripening. Antioxidant activity was screened with electron spin resonance spectrometry and trolox equivalent antioxidant capacity (TEAC) assay. An adequate picture of phenolic compounds developed during maturation and ripening was determined using HPLC-DAD. Anthocyanins of all varieties increased during successive harvest stages; meanwhile flavonols and hydroxycinnamic acids decreased from unripe green to ripe blue stage of berry ripening. Blueberry antioxidant activity, as well as total phenolic content tended to decrease during ripening.

In many microorganisms, fungi and algae (1→3), (1→6)-β- d -glucan can be found as cell wall polysaccharide. Various publications describe the strong positive influence of these glucans on the immune system comprising antibacterial, wound-healing and antitumour activities. Recently, it was found that (1→3)-β- d -glucan from the yeast Saccharomyces cerevisiae also exhibits antioxidative capabilities. In the present study, the antioxidative activity of glucan from the cell walls of S. cerevisiae grown on different media (wort, yeast–peptone–glucose, yeast–peptone–galactose) was investigated. Fermentation of the yeast took place on three different media. After fermentation the yeast was harvested and disrupted in a high-pressure homogeniser. Subsequently, glucan was extracted from the yeast cell walls by a procedure including a combination of hot water and enzymatic treatment. The level of (1→3), (1→6)-β- d -glucan in the cell walls was analysed enzymatically. The antioxidant activity was determined by electron paramagnetic resonance (EPR) spectrometry, TEAC assay (Trolox equivalent antioxidant capacity) and an ‘amperometric biosensor’. The results show significant differences in the glucan content of the cell walls and in their antioxidative activities depending on growth medium. However, glucan itself seems to have a low antioxidative activity in contrast to other cell wall fractions e.g. proteins.

The aim of the present study was to test whether variations in the root environment affect the content of health-related organosulfur compounds, total phenolic compounds, and flavonol glycoside concentrations in onions. For this purpose, greenhouse-grown onions ( Allium cepa L.) were either inoculated with a commercial arbuscular mycorrhizal inoculum or a sterile inoculum and were provided with two NH(4)(+):NO(3)(-) ratios as a nitrogen source. Onion growth, arbuscular mycorrhizal colonization rate, sugars, and nutrient element concentrations were also quantified. The plant antioxidant activity and quercetin monoglucoside and organosulfur compound concentrations increased with dominant nitrate supply. Furthermore, mycorrhizal colonization increased the antioxidant activity and also concentrations of the major quercetin glucosides. The present study provides clear evidence that antioxidant activity, quercetin glycosides, and organosulfur compounds can be increased in sufficiently supplied onion plants by dominant nitrate supply or application of arbuscular mycorrhizal fungi. This was probably due to increased precursor production and induced defense mechanisms.

(1-->3),(1-->6)-Beta-D-Glucan, a cell wall polysaccharide in many microorganisms, fungi and algae, is a well-known biological response modifier. Recently, it was found that (1-->3)-beta-D-glucan from Saccharomyces cerevisiae also exhibits antioxidative capabilities. In this study the antioxidative activity of the cell wall fractions of brewer's yeast was investigated. Particular emphasis was put on the question to which extent glucan is responsible for the antioxidative activity of the cell walls and how the other cell wall components might contribute. For the experiments yeast cell walls from brewery fermentations were used. Glucan was isolated by a three-step extraction procedure including a combination of hot water and enzymatic treatment. The level of (1-->3),(1-->6)-beta-D-glucan in the cell walls was analyzed enzymatically. The antioxidant activity was determined by electron paramagnetic resonance spectrometry and Trolox equivalent antioxidant capacity assay. The results show that the antioxidative activity of yeast cell wall proteins exceeds that of beta-glucan greatly. Especially aromatic side chains and free thiols from denatured proteins seem to work as antioxidants.

Flavonoids are an important constituent of the human diet. In recent years, they have gained much attention due to their physiological properties, leading to an enormous increase in research on cancer prevention and reduction of cardiovascular diseases. Unfortunately, there is limited information about the fate of flavonoid glycosides during thermal treatment such as cooking, frying, roasting, etc. Such processing techniques may have an impact on the flavonoid structure, resulting in changes of the bioavailability and activity of the flavonoids. In this study, the stability of selected model and onion quercetin glycosides under roasting conditions (180 degrees C) was determined. The influence of the kind and position of the sugar moiety was investigated. As onions contain large amounts of quercetin glycosides and are often subject to thermal processes in food production, their major glycosides were isolated using counter current chromatography and roasted. The thermal treatment led to a degradation of the quercetin glycosides. The main product is the aglycone quercetin, which remained stable during further roasting. During the roasting process of the quercetin diglucoside isolated from onion, the formation of a monoglycoside as an intermediate product was observed. This underlined that the stability of the glycosides is dependent on the kind and position of the sugar moiety.

A novel method for the determination of phomopsin A (1) in lupin flour, pea flour, and bean flour as well as whole lupin plants was established based on stable isotope dilution assay (SIDA) LC-MS/MS using (15)N6-1 as an isotopically labeled internal standard. Artificially infected samples were used to develop an optimized extraction procedure and sample pretreatment. The limits of detection were 0.5-1 μg/kg for all matrices. The limits of quantitation were 2-4 μg/kg. The method was used to analyze flour samples generated from selected legume seeds and lupin plant samples that had been inoculated with Diaporthe toxica and two further fungal strains. Finally, growing lupin plants infected with D. toxica were investigated to simulate a naturally in-field mycotoxicosis. Toxin levels of up to 10.1 μg/kg of 1 were found in the pods and 7.2 μg/kg in the stems and leaves.

In Press, Unroasted cocoa beans are rich in monomeric flavanols and particularly epicatechin-based proanthocyanidin oligomers, with the latter making up to 60% of the total polyphenol content. Although the antioxidant activity of cocoa polyphenols is well known, it is still a challenging analytical field, especially, when it comes to the determination of condensed polyphenols and the evaluation of their single contribution to the overall activity. Therefore, an online NP-HPLC-DPPH assay was developed to separate the homologous series of condensed polyphenols for assessing their antioxidant capacity in relation to their degree of polymerization (DP), simultaneously. In this context, normal-phase chromatography allows separation of polyphenols based on their degree of polymerization. This study showed that an unroasted cocoa extract contains condensed polyphenols with a DP of up to 10 monomer units. By means of the online post-column derivatisation with 2,2-diphenyl-1-picrylhydrazyl (DPPH), the antioxidant capacity of the separated condensed polyphenols was assessed. It could be shown that, with the exception of the dimers, the contribution to the total antioxidant activity decreased from monomers to decamers. However, from the single proanthocyanidins identified, nonameric and decameric proanthocyanidins were found to have the highest values for the antioxidant capacity. The degree of polymerization associated with each molecular weight fraction was further confirmed using electrospray ionization mass spectrometry coupled with reverse-phase liquid chromatography. The online NP-HPLC-DPPH method can be used as qualitative and quantitative analysis of condensed proanthocyanidins and the simultaneous elucidation of the biological activity of proanthocyanidins in complex mixtures.

Chemoprotective or genotoxic effects of glucosinolates occurring in Brassica vegetables are attributed to their hydrolysis products formed upon tissue damage by plant myrosinase. Since Brassica vegetables, in which myrosinase has been heat-inactivated, still display bioactivity, glucosinolate activation has been attributed to intestinal bacteria. The aim of this study was to investigate whether this is true. Glucoraphanin (172 mg/kg body weight) and neoglucobrassicin (297 mg/kg body weight) were administered intragastrically to germ free and human microbiota associated (HMA) mice. Approximately 30% of the applied doses of glucoraphanin and neoglucobrassicin were excreted unchanged in the urine of both germ free and HMA mice. Isothiocyanates, sulforaphane, and erucin, formed from glucoraphanin, were mainly excreted as urinary N-acetyl-l-cysteine conjugates. N-Methoxyindole-3-carbinol formed from neoglucobrassicin was observed in small amounts in both germ free and HMA mice. Formation of DNA adducts from neoglucobrassicin was also independent from bacterial colonization of the mice. Hence, intestinal bacteria are involved in the bioactivation of glucosinolates in the gut, but their contribution to glucosinolate transformation in HMA mice is apparently very small.

This study aimed at developing a rapid chromatographic assay to monitor phosphorylation sites in peptides. For the analysis of nociceptive signal transduction pathways, the detection of phosphorylated proteins/peptides plays a fundamental role. To get further insights in the phosphorylation mechanism of protein kinase C-e (PKC-e) and protein kinase A (PKA), potential targets were divided into subsections resulting in peptides that contain only one possible phospho-binding site. The use of high-performance thin-layer chromatography (HPTLC) offers the possibility of a high throughput of samples and the advantage of a quick sample clean-up. A combined strategy of an effect-directed overlay procedure on the TLC plate using specific antibodies (immunostaining, HPTLC-IS) as well as a parallel, direct mass spectrometric methodology by HPTLC-MALDI-TOF-MS was developed. With regard to HPTLC-IS, validation of the data exhibited a lower limit of detection than the traditionally used protein derivatization reagent fluorescamine. Besides the identification of the phosphorylated peptides, a semi-quantitative estimation can be performed with HPTLC-IS.

In recent years, protein chemistry tends inexorably toward the analysis of more complex proteins, proteoforms, and posttranslational protein modifications. Although mass spectrometry developed quite fast correspondingly, sample preparation and separation of these analytes is still a major issue and quite challenging. For many years, electrophoresis seemed to be the method of choice; nonetheless its variance is limited to parameters such as size and charge. When taking a look at traditional (thin-layer) chromatography, further parameters such as polarity and different mobile and stationary phases can be utilized. Further, possibilities of detection are manifold compared to electrophoresis. Similarly, two-dimensional separation can be also performed with thin-layer chromatography (TLC). As the revival of TLC developed enormously in the last decade, it seems to be also an alternative to use high performance thin-layer chromatography (HPTLC) for the separation of proteins. The aim of this study was to establish an HPTLC separation system that allows a separation of protein mixtures over a broad polarity range, or if necessary allowing to modify the separation with only few steps to improve the separation for a specific scope. Several layers and solvent systems have been evaluated to reach a fully utilized and optimized separation system.

Cancer-cell heterogeneity dramatically influences treatment success, but escapes detection by classical histology. Mass-spectrometric imaging (MSI) represents a powerful method for visualizing the spatial distribution of proteins in tissue sections. Herein we asked whether MSI also facilitates detection of tumor heterogeneity. We first transduced the human neuroendocrine-carcinoma BON cell line following the red-green-blue (RGB) marking principle. RGB marking allows for specific color-coding of individual clones. Mice transplanted with RGB-marked BON cells developed liver tumors. We identified 16 primary tumors clearly distinguishable by histology and fluorescence imaging, but also based on a common tumor-specific signal pattern detected by MSI. Importantly, this pattern was clearly confined to tumor tissue while was absent from surrounding liver tissue. At the same time, we observed protein signals differentially present in a few or even single tumors. Since these signals were independent of RGB marking, they apparently reflected unique intrinsic protein-signal patterns of individual tumors. Thus, our data propose MSI as a tool for identifying divergent tissue by 'fingerprints' of protein signals, allowing not only for differentiation of tumor from healthy tissue but also detection of tumor heterogeneity. In conclusion, by visualizing tumor heterogeneity, MSI ideally complements microscopy-based methods. This might help to better understand tumor biology and develop future treatment strategies.

Minimally processed fruit products such as smoothies are increasingly coming into demand. However, they are often combined with dairy ingredients. In this combination, phenolic compounds, polyphenoloxidases, and amino compounds could interact. In this work, a model approach is presented where apple serves as a source for a high polyphenoloxidase activity for modulating the reactions. The polyphenoloxidase activity ranged from 128 to 333 nakt/mL in different apple varieties. From these, ‘Braeburn’ was found to provide the highest enzymatic activity. The formation and stability of resulting chromogenic conjugates was investigated. The results show that such adducts are not stable and possible degradation mechanisms leading to follow-up products formed are proposed. Finally, apple extracts were used to modify proteins and their functional properties characterized. There were retaining antioxidant properties inherent to phenolic compounds after adduct formation. Consequently, such interactions may also be utilized to improve the textural quality of food products.

Scope Glucosinolates are secondary metabolites present in Brassica vegetables. Alkenyl glucosinolates are enzymatically degraded forming nitriles or isothiocyanates, but in the presence of epithiospecifier protein, epithionitriles are released. However, studies on the occurrence of epithionitriles in Brassica food and knowledge about their biological effects are scarce. Methods and results Epithionitrile formation from glucosinolates of seven Brassica vegetables was analyzed using GC-MS and HPLC-DAD. Bioactivity of synthetic and plant-derived 1-cyano-2,3-epithiopropane (CETP) – the predominant epithionitrile in Brassica vegetables – in three human hepatocellular carcinoma (HCC) cell lines and primary murine hepatocytes was also evaluated. The majority of the Brassica vegetables were producers of nitriles or epithionitriles as hydrolysis products and not of isothiocyanates. For example, Brussels sprouts and savoy cabbage contained up to 0.8 μmol CETP/g vegetable. Using formazan dye assays, concentrations of 380–1500 nM CETP were observed to inhibit the mitochondrial dehydrogenase activity of human HCC cells without impairment of cell growth. At 100-fold higher CETP concentrations, cell death was observed. Presence of plant matrix increased CETP-based toxicity. Conclusion These in vitro data provide no indication that epithionitriles will severely affect human health by Brassica consumption. In contrast to isothiocyanates, no evidence of selective toxicity against HCC cells was found.

The chemistry of glucosinolates and their behavior during food processing is very complex. Their instability leads to the formation of a bunch of breakdown and reaction products that are very often reactive themselves. Although excessive consumption of cabbage varieties has been thought for long time to have adverse, especially goitrogenic effects, nowadays, epidemiologic studies provide data that there might be beneficial health effects as well. Especially Brassica vegetables, such as broccoli, radish, or cabbage, are rich in these interesting plant metabolites. However, information on the bioactivity of glucosinolates is only valuable when one knows which compounds are formed during processing and subsequent consumption. This review provides a comprehensive, in-depth overview on the chemical reactivity of different glucosinolates and breakdown products thereof during food preparation.

Currently, there is great momentum in research on phytochemicals, such as polyphenols, in fruit and vegetables. In this context, berry crops have drawn much attention. Blueberries score extraordinarily well in rankings among polyphenol-rich berry fruits. For blueberries, research has focused primarily on the phenolic compound profiles and antioxidant activities. However, little is reported on the development of phenolic compounds during the process of fruit maturation and ripening as well as during post-harvest. Maturation and ripening of blueberries are accompanied by characteristic physiological changes, which include color change, tissue softening, increasing maturity index. etc. During the different physiological stages shifting within the phenolic compound profile occurs as follows: anthocyanins increase during successive ripening, meanwhile flavonols and hydroxycinnamic acids decrease from unripe green to ripe blue berries. Depending on the harvest date, this trend continues during post-harvest. However, while earlier-harvested berries continue phenol accumulation, all phenolic compounds are degraded in stored full-ripe berries.

Keywords: Antioxidative activity; Antioxidative capacity; Electron spin resonance spectroscopy; Plant phenolic compounds; Radicals

Bromelain was allowed to react with phenolic compounds. The activity and selected physico-chemical properties of the resulting derivatives were characterized. In vitro experiments showed that the proteolytic activity of bromelain was inhibited. Bromelain also serves as a food protein, because food stuffs based on pineapple contain relatively high concentrations of bromelain. In vitro digestion of bromelain derivatives with the main proteolytic enzymes of the gastrointestinal tract was also adversely affected. A covalent attachment of the phenolic compounds was identified at the tryptophan, free amino (lysines and N-terminal) and thiol groups of bromelain. A decrease in solubility of the derivatives was observed. The isoelectric point was shifted to lower pH values and high molecular weight fractions were identified. All effects observed depended on the reactivity of the phenolic substances. Two supplementary food products containing both bromelain and quercetin were also tested in terms of their proteolytic activity and digestibility.

During processing and storage, phenolic compounds (PCs) may react with food protein bound amino acids (AAs). Such reactions have been reported to change physicochemical and to decrease in vitro digestion properties of proteins. A rat growth and nitrogen (N) balance study was conducted to prove whether derivatization with chlorogenic acid (CA) affects the nutritional quality of beta-lactoglobulin (beta-LG). Test diets (10% protein level) contained nonderivatized beta-LG (LG, treated under omission of CA), low derivatization level beta-LG (LGL), high derivatization level beta-LG (LGH), or casein supplemented with l-methionine (0.3% of diet; C+met) as an internal standard. An additional group received untreated beta-LG supplemented with pure CA (1.03% of diet; LG+CA). The AA composition of test proteins, plasma AAs, and liver glutathione (GSH) concentrations were determined. Protein digestibility-corrected amino acid score (PDCAAS) was calculated using human or rat AA requirement patterns and rat fecal digestibility values. N excretion was significantly higher in feces and lower in urine of rats fed with LGH as compared to LG and LGL. Consequently, true N digestibility (TND) was significantly lower with LGH as compared to LG and LGL. The lower content of methionine, cysteine, lysine, and tryptophan in LGH corresponded to a reduced TND. Net protein utilization (NPU) was not different between treated beta-LG fed diet groups but was lower than in LG+CA and C+met fed groups. Only at a relatively high level of derivatization with CA, the otherwise good nutritional quality of beta-LG is affected so that TND is reduced, while NPU still remains unaffected. Derivatization of beta-LG with CA does not seem to lead to an additional deficiency in a specific indispensable AA in growing rats fed with 10% protein.

Bovine serum albumin (BSA) was derivatized by covalent attachment of different amounts of quercetin [ratios of BSA to quercetin were 20:1, 10:1, 7:1, 5:1, and 2:1 (w/w)]. The antioxidant activity of the protein-phenol derivatives was investigated using a modified TEAC assay. The results show that the covalent attachment of quercetin to BSA decreases the total antioxidant activity in comparison to an equivalent amount of free quercetin depending on the degree of derivatization. The derivative with the highest amount of covalently bound quercetin (the 2:1 derivative) showed an antioxidant activity of only 79% compared to an equivalent amount of free quercetin. After the enzymatic proteolysis of the BSA-quercetin derivatives with trypsin, the total antioxidant activity of the degradation products increases in comparison to the respective undigested derivatives but does not reach the activity of an equivalent amount of free quercetin. Even after 240 min of tryptic degradation, there is still a lack in antioxidant activity (for the 7:1 derivative nearly 33%) as compared to free quercetin.

Secondary plant metabolites are important native food components, which are becoming more and more interesting due to their physiological effects on human beings. One of the largest groups of these compounds is represented by plant phenols. This review summarizes the structure, classification and distribution of the phenolic compounds in plant foods, their chemistry and signification with regard to food processing and -storage as well as their physiological effects. This work focuses mainly on such reactions of the phenolic substances with proteins and enzymes that lead to covalent bonds. The derivatives formed have been characterized in terms of changes in their physicochemical and structural properties. The effect on the proteolytic in vitro digestion has been also illustrated. Further aspects reported include the influence on enzyme activity and -kinetic parameters. The different aspects of the nutritional-physiological consequences of such reactions in food and body, especially considering their significance to food science and technology are discussed.

Selected enzymes (alpha-amylase, trypsin, and lysozyme) were allowed to react with some simple phenolic and related compounds (caffeic acid, chlorogenic acid, ferulic acid, gallic acid, m-, o-, and p-dihydroxybenzenes, quinic acid, and p-benzoquinone). The derivatized enzymes obtained were characterized in terms of their activity. In vitro experiments showed that the enzymatic activity of the derivatives was adversely affected. This enzyme inhibition depended on the reactivity of the phenolic and related substances tested as well as on the kind of substrate applied. The decrease in the activity was accompanied by a reduction in the amount of free amino and thiol groups, as well as tryptophan residues, which resulted from the covalent attachment of the phenolic and related compounds to these reactive nucleophilic sites in the enzymes. The enzyme inhibition correlates well with the blocking of the mentioned amino acid side chains.

Scope Nasturtium plants contain the glucosinolate glucotropaeolin and its corresponding breakdown product benzyl isothiocyanate (BITC), the latter being intensively studied with regard to cancer chemoprevention and anti-inflammatory properties. In addition, recent research has shown that isothiocyanates are able to activate the release of several gut hormones in vitro and in rodent studies. Here, we tested the effects of a dietary nasturtium administration on circulating levels of gut hormones in humans. Methods and results Metabolically healthy males (n = 15) received a single oral dose of 10 g freeze-dried nasturtium leaf material suspended in water or only water (control). Blood samples were taken every hour and serum concentrations of insulin, C-peptide, glucagon-like peptide 1 (GLP-1), glucose-dependent insulinotropic peptide (GIP), and peptide (PYY) were analyzed. Oral nasturtium intake resulted in an increased release of PYY over a time period of 6 h whereas circulating levels of other hormones were not changed. Conclusion Given the finding that nasturtium consumption enhances secretion of PYY, a key hormone involved in energy regulation, special diets containing nasturtium, or supplementation with nasturtium or BITC might be considered in the treatment of obesity.

Background: Ethiopian kale (Brassica carinata) is a horticulturally important crop used as leafy vegetable in large parts of East and Southern Africa. The leaves are reported to contain high concentrations of health-promoting secondary plant metabolites. However, scientific knowledge on their health benefits is scarce.Objective: This study aimed to determine the cancer preventive potential of B. carinata using a human liver in vitro model focusing on processing effects on the pattern of secondary plant metabolites and bioactivity.Design: B. carinata was cultivated under controlled conditions and differentially processed (raw, fermented, or cooked) after harvesting. Human liver cancer cells (HepG2) were treated with ethanolic extracts of raw or processed B. carinata leaves and analyzed for their anti-genotoxic, anti-oxidant, and cytostatic potential. Chemical analyses were carried out on glucosinolates including breakdown products, phenolic compounds, carotenoids, and chlorophyll content.Results: P...

The food-related isothiocyanate sulforaphane (SFN), a hydrolysis product of the secondary plant metabolite glucoraphanin, has been revealed to have cancer-preventive activity in experimental animals. However, these studies have often provided inconsistent results with regard to bioavailability, bioaccessibility, and outcome. This might be because the endogenous biotransformation of SFN metabolites to the structurally related erucin (ERN) metabolites has often not been taken into account. In this work, a fully validated liquid chromatography tandem mass spectrometry (LC-MS-MS) method was developed for the simultaneous determination of SFN and ERN metabolites in a variety of biological matrices. To reveal the importance of the biotransformation pathway, matrices including plasma, urine, liver, and kidney samples from mice and cell lysates derived from colon-cancer cell lines were included in this study. The LC-MS-MS method provides limits of detection from 1 nmol L(-1) to 25 nmol L(-1) and a mean recovery of 99 %. The intra and interday imprecision values are in the range 1-10 % and 2-13 %, respectively. Using LC-MS-MS, SFN and ERN metabolites were quantified in different matrices. The assay was successfully used to determine the biotransformation in all biological samples mentioned above. For a comprehensive analysis and evaluation of the potential health effects of SFN, it is necessary to consider all metabolites, including those formed by biotransformation of SFN to ERN and vice versa. Therefore, a sensitive and robust LC-MS-MS method was validated for the simultaneous quantification of mercapturic-acid-pathway metabolites of SFN and ERN.