Bioactivity of Ruta graveolens and Satureja montana Essential Oils on Solanum tuberosum Hairy Roots and Solanum tuberosum Hairy Roots with Meloidogyne chitwoodi Co-cultures.

As a nematotoxics screening biotechnological system, Solanum tuberosum hairy roots (StHR) and S. tuberosum hairy roots with Meloidogyne chitwoodi co-cultures (StHR/CRKN) were evaluated, with and without the addition of the essential oils (EOs) of Satureja montana and Ruta graveolens. EOs nematotoxic and phytotoxic effects were followed weekly by evaluating nematode population density in the co-cultures as well as growth and volatile profiles of both in vitro cultures types. Growth, measured by the dissimilation method and by fresh and dry weight determination, was inhibited after EO addition. Nematode population increased in control cultures, while in EO-added cultures numbers were kept stable. In addition to each of the EOs main components, and in vitro cultures constitutive volatiles, new volatiles were detected by gas chromatography and gas chromatography coupled to mass spectrometry in both culture types. StHR with CRKN co-cultures showed to be suitable for preliminary assessment of nematotoxic EOs.

In vitro co-cultures of Pinus pinaster with Bursaphelenchus xylophilus: a biotechnological approach to study pine wilt disease.

MAIN CONCLUSION: Co-cultures of Pinus pinaster with Bursaphelenchus xylophilus were established as a biotechnological tool to evaluate the effect of nematotoxics addition in a host/parasite culture system. The pinewood nematode (PWN), Bursaphelenchus xylophilus, the causal agent of pine wilt disease (PWD), was detected for the first time in Europe in 1999 spreading throughout the pine forests in Portugal and recently in Spain. Plant in vitro cultures may be a useful experimental system to investigate the plant/nematode relationships in loco, thus avoiding the difficulties of field assays. In this study, Pinus pinaster in vitro cultures were established and compared to in vivo 1 year-old plantlets by analyzing shoot structure and volatiles production. In vitro co-cultures were established with the PWN and the effect of the phytoparasite on in vitro shoot structure, water content and volatiles production was evaluated. In vitro shoots showed similar structure and volatiles production to in vivo maritime pine plantlets. The first macroscopic symptoms of PWD were observed about 4 weeks after in vitro co-culture establishment. Nematode population in the culture medium increased and PWNs were detected in gaps of the callus tissue and in cavities developed from the degradation of cambial cells. In terms of volatiles main components, plantlets, P. pinaster cultures, and P. pinaster with B. xylophilus co-cultures were all ß- and α-pinene rich. Co-cultures may be an easy-to-handle biotechnological approach to study this pathology, envisioning the understanding of and finding ways to restrain this highly devastating nematode.

Bioactivity against Bursaphelenchus xylophilus: Nematotoxics from essential oils, essential oils fractions and decoction waters.

The Portuguese pine forest has become dangerously threatened by pine wilt disease (PWD), caused by the pinewood nematode (PWN), Bursaphelenchus xylophilus. Synthetic chemicals are the most common pesticides used against phytoparasitic nematodes but its use has negative ecological impacts. Phytochemicals may prove to be environmentally friendly alternatives. Essential oils (EOs) and decoction waters, isolated from 84 plant samples, were tested against B. xylophilus, in direct contact assays. Some successful EOs were fractionated and the fractions containing hydrocarbons or oxygen-containing molecules tested separately. Twenty EOs showed corrected mortalities ⩾96% at 2 µL/mL. These were further tested at lower concentrations. Ruta graveolens, Satureja montana and Thymbra capitata EOs showed lethal concentrations (LC100)<0.4µL/mL. Oxygen-containing molecules fractions showing corrected mortality ⩾96% did not always show LC100 values similar to the corresponding EOs, suggesting additive and/or synergistic relationships among fractions. Nine decoction waters (remaining hydrodistillation waters) revealed 100% mortality at a minimum concentration of 12.5µL/mL. R. graveolens, S. montana and T. capitata EOs are potential environmentally friendly alternatives for B. xylophilus control given their high nematotoxic properties. Nematotoxic activity of an EO should be taken in its entirety, as its different components may contribute, in distinct ways, to the overall EO activity.

First study on antimicriobial activity and synergy between isothiocyanates and antibiotics against selected Gram-negative and Gram-positive pathogenic bacteria from clinical and animal source.

The emergence of new diseases and the resurgence of several infections that were controlled in the past, associated with recent increase of bacterial resistance have created the necessity for more studies towards to the development of new antimicrobials and new treatment strategies. The aim of the present study was to evaluate the in vitro synergy between different classes of important glucosinolates hydrolysis products-isothiocyanates with antibiotics (gentamycin and vancomycin), against important pathogenic bacteria: Escherichia coli, Enterococcus faecalis, Listeria monocytogenes, Pseudomonas aeruginosa and Staphylococcus aureus. A disc diffusion method was used to evaluate the antibacterial activity. The antimicrobial activity of phytochemicals and combinations between gentamycin, vancomycin and phytochemicals were quantitatively assessed by measuring the inhibitory halos. The results showed a selective antimicrobial effect of isothiocyanates, and this effect was strictly related with their chemical structure. In general the benzylisothiocyanate was the most effective compound against both Gram-positive and Gram-negative bacteria. The Listeria monocytogenes and Staphylococcus aureus were the bacteria most affected either by the phytochemicals alone or by the combination phytochemical-antibiotic. The bacteria Pseudomonas aeruginosa was the less affected pathogen. The most important synergism detected occurred between the commercial antibiotics with benzylisothiocyanate and 2-phenylethylisothiocyanate. In conclusion, some isothiocyanates are effective inhibitors of in vitro bacterial growth, and they can act synergistically with antibiotics.

Antibacterial effects of glucosinolate-derived hydrolysis products against enterobacteriaceae and enterococci isolated from pig ileum segments.

The aim of this study was to evaluate the effects of various glucosinolate-derived hydrolysis products (HP) as antibacterial compounds against Enterobacteriaceae and Enterococcaceae isolated from intestinal segments of healthy pigs collected directly from slaughter-houses in the North of Portugal. Using a previously described disk-diffusion bioassay, all HP were tested at six different doses (0.015, 0.15, 0.75, 1.5, 3.0, and 15.0 µmoles) in dimethyl-sulfoxide (DMSO), with the exception of sulforaphane (SFN), which was not tested at 15.0 µmoles. Positive (antibiotic standard) and negative controls (DMSO) were included in all experiments. All the experiments were conducted in triplicate. In vitro inhibition of the bacterial growth by the HP was proportional to the concentration used and in many cases was even higher than for the gentamycin, the antibiotic control. The results clearly showed that the glucosinolates-derived HPs were very effective in vitro inhibitors of bacterial growth. The natural products, and specifically the isothiocyanates, should be evaluated as potential alternative control agents for potentially pathogenic bacteria (e.g., dietary amendment of pig foods with glucosinolate-containing plants).

Seasonal effects on bioactive compounds and antioxidant capacity of six economically important brassica vegetables.

Research on natural and bioactive compounds is increasingly focused on their effects on human health, but there are unexpectedly few studies evaluating the relationship between climate and natural antioxidants. The aim of this study was analyze the biological role of six different Brassica vegetables (Brassica oleracea L. and Brassica rapa L.) as a natural source of antioxidant compounds. The antioxidant activity may be assigned to high levels of L-ascorbic acid, total phenolics and total flavonoids of each sample. The climate seasons affected directly the concentration of bioactive components and the antioxidant activity. Broccoli inflorescences and Portuguese kale showed high antioxidant activity in Spring-Summer whilst turnip leaves did so in Summer-Winter. The Brassica vegetables can provide considerable amounts of bioactive compounds and thus may constitute an important natural source of dietary antioxidants.

Effect of cooking on total vitamin C contents and antioxidant activity of sweet chestnuts (Castanea sativa Mill.).

In this work the total vitamin C contents (ascorbic acid+dehydroascorbic acid) and antioxidant activity of raw and cooked chestnuts was evaluated. The vitamin C contents of raw chestnuts varied significantly between the different cultivars (cv) studied and it varied from 400mg/kg dry weight (cv Lada) to 693mg/kg dry weight (cv Martaínha). The different cultivars behave differently during the cooking process concerning the loss of vitamin C. A significant decrease in the vitamin C content of the chestnuts was observed, 25-54% for the boiling process and 2-77% for the roasting process. Boiled and roasted chestnuts can be good sources of vitamin C since it may represent 22.4%, 16.2%, 26.8% and 19.4%, respectively, of the recommended dietary intake for an adult man and woman. The cooking process significantly changed the antioxidant activity of the chestnuts. A difference was observed between the cultivars during the cooking processes, concerning the antioxidant activity. For the raw chestnuts the variation in vitamin C content of the chestnuts explains 99% of the antioxidant activity variation but for the roasted and boiled chestnuts this percentage significantly decreases to 51% and 88%, respectively. Although a high antioxidant activity is still present in the cooked chestnuts, the cause for this antioxidant activity is less dependent on the vitamin C content of the chestnuts, probably due to the conversion of ascorbic acid to dehydroascorbic acid. The increase in gallic acid during the cooking process, presumably transferred from the peels to the fruit, also contributes to the high antioxidant activity observed for the cooked chestnuts.

Antimicrobial activity of phenolics and glucosinolate hydrolysis products and their synergy with streptomycin against pathogenic bacteria.

The purpose of the present study was to evaluate the in vitro antibacterial effects of different classes of important and common dietary phytochemicals (5 simple phenolics - tyrosol, gallic acid, caffeic acid, ferulic acid, and chlorogenic acid; chalcone - phloridzin; flavan-3-ol - (-) epicatechin; seco-iridoid - oleuropein glucoside; 3 glucosinolate hydrolysis products - allylisothiocyanate, benzylisothiocyanate and 2-phenylethylisothiocyanate) against Escherichia coli, Pseudomonas aeruginosa, Listeria monocytogenes and Staphylococcus aureus. Another objective of this study was to evaluate the effects of dual combinations of streptomycin with the different phytochemicals on antibacterial activity. A disc diffusion assay was used to evaluate the antibacterial activity of the phytochemicals and 3 standard antibiotics (ciprofloxacin, gentamicin and streptomycin) against the four bacteria. The antimicrobial activity of single compounds and dual combinations (streptomycin-phytochemicals) were quantitatively assessed by measuring the inhibitory halos. The results showed that all of the isothiocyanates had significant antimicrobial activities, while the phenolics were much less efficient. No antimicrobial activity was observed with phloridzin. In general P. aeruginosa was the most sensitive microorganism and L. monocytogenes the most resistant. The application of dual combinations demonstrated synergy between streptomycin and gallic acid, ferulic acid, chlorogenic acid, allylisothiocyanate and 2-phenylethylisothiocyanate against the Gram-negative bacteria. In conclusion, phytochemical products and more specifically the isothiocyanates were effective inhibitors of the in vitro growth of the Gram-negative and Gram-positive pathogenic bacteria. Moreover, they can act synergistically with less efficient antibiotics to control bacterial growth.

Phenolics and antioxidant properties of fruit pulp and cell wall fractions of postharvest banana (Musa acuminata Juss.) cultivars.

Banana fruits are important foods, but there have been very few studies evaluating the phenolics associated with their cell walls. In the present study, (+) catechin, gallocatechin, and (-) epicatechin, as well as condensed tannins, were detected in the soluble extract of the fruit pulp; neither soluble anthocyanidins nor anthocyanins were present. In the soluble cell wall fraction, two hydroxycinnamic acid derivatives were predominant, whereas in the insoluble cell wall fraction, the anthocyanidin delphinidin, which is reported in banana cell walls for the first time, was predominant. Cell wall fractions showed remarkable antioxidant capacity, especially after acid and enzymatic hydrolysis, which was correlated with the total phenolic content released after the hydrolysis of the water-insoluble polymer, but not for the posthydrolysis water-soluble polymer. The acid hydrolysis released various monosaccharides, whereas enzymatic hydrolysis released one peak of oligosaccharides. These results indicate that banana cell walls could be a suitable source of natural antioxidants and that they could be bioaccessible in the human gut.

Composition of European chestnut (Castanea sativa Mill.) and association with health effects: fresh and processed products.

Chestnut fruits are highly regarded and widely consumed throughout Europe, America and Asia. Various commercial forms are available, e.g. fresh and industrially processed. There have been various reviews on the composition of chestnut fruits but there has not been a comprehensive review of the different health benefits that this fruit can provide. This review is focused on the composition and associated health effects of European fresh chestnut (Castanea sativa Mill.) fruits and their home-processed and industrial products, e.g. boiled, roasted, frozen, and 'marron glacées'. We also expand the knowledge of chestnut uses by presenting data for other chestnut materials that have potential applications as new foods, as sources of antioxidants, and as sources of other useful bioactives. There is considerable literature data on nutrients in fresh chestnut fruits but less information on bioactive non-nutrients such as phenolics. Chestnuts are mostly consumed as processed forms, and the different types of processing clearly affect the nutrient and non-nutrient composition of the fruits. The benefits that this fruit can provide for human and animal health are numerous, but it is clear that improvements can be made for both production and quality of chestnut products, e.g. genetic selection and optimizing industrial processing.

Anatomical, chemical, and biochemical characterization of cladodes from prickly pear [Opuntia ficus-indica (L.) Mill.].

Opuntia ficus-indica cladodes represent the green stem of the plant and are generally used as animal feed or disposed of in landfills. The present work investigated the anatomical and chemical composition of Opuntia cladodes, which form the basis of their pharmacological effects. Glucose and galacturonic acid were the main sugars of Opuntia cladodes, whereas high-performance liquid chromatography (HPLC) analysis showed the presence of mainly kaempherol and isorhamnetin glycosides (glucoside and rhamnoside). The presence of high amounts of calcium oxalate crystals was demonstrated by light microscopy on fresh and lyophilized cladodes. No antimicrobial activity was observed even after enzymatic treatment. O. ficus-indica cladodes may retain material tightly associated with cell-wall components, and this property will have the potential to greatly reduce the bioavailability of bioactive compounds.

Absorption, Conjugation and Efflux of the Flavonoids, Kaempferol and Galangin, Using the Intestinal CACO-2/TC7 Cell Model.

Flavonoids are biologically active compounds in food with potential health effects. We have used the Caco-2 cell monolayer model to study the absorption and metabolism of two flavonols, a class of flavonoids, specifically kaempferol and galangin. Metabolism experiments allowed identification of 5 kaempferol conjugates: 3-, 7- and 4'-glucuronide, a sulphate and a glucurono-sulphate; and 4 galangin conjugates: 3-, 5- and 7-glucuronides, and a sulphate, using specific enzyme hydrolysis, HPLC-MS, and HPLC with post column metal complexation/tandem MS. Transport studies showed that the flavonols were conjugated inside the cells then transported across the monolayer or effluxed back to the apical side. Sulphated conjugates were preferentially effluxed back to the apical side, whereas glucuronides were mostly transported to the basolateral side. For kaempferol, a small amount of the unconjugated aglycone permeated in both directions, indicating some passive diffusion. When kaempferol-3-glucuronide and quercetin7-sulphate were applied to either side of the cells, no permeation in either direction was observed, indicating that conjugates cannot re-cross the cell monolayer. Formation of apical kaempferol-7- and 4'-glucuronides was readily saturated, whereas formation of other conjugates at the apical side and all at the basolateral side increased with increasing concentration of kaempferol, implying different transporters are responsible at the apical and basolateral sides. The results highlight the important but complex metabolic changes occurring in flavonoids during absorption.

Polyamine metabolism and transforming growth factor-beta signaling are affected in Caco-2 cells by differentially cooked broccoli extracts.

The health benefits of consuming cruciferous vegetables are widely considered to be due to the biological activity of glucosinolate degradation products. However, it is conceivable that other phytochemicals within crucifers may also have biological activity that may contribute to health benefits. In this study, we analyzed global gene expression in Caco-2 cells exposed to extracts derived from broccoli that had been heat treated to different extents to result in contrasting profiles of glucosinolates and their degradation products. Extracts microwaved for 0, 1, and 4 min contained 9.5, 25.5, and 0 micromol/L sulforaphane and induced changes in expression of 381, 1017, and 101 genes, respectively (>2 fold; P < 0.01). Seventy-two genes showed similar changes in expression after treatment with all 3 extracts. These included genes involved in polyamine catabolism and transforming growth factor (TGF)-beta signaling. Consistent with these changes in gene expression, subsequent studies demonstrated that exposing cells to these extracts, including the 4-min extract that contained no glucosinolate degradation products, increased putrescine and N-acetyl-spermine concentration, and suppressed the TGFbeta1-mediated induction of phosphorylated Smad 2. This is the first report, to our knowledge, of phytochemicals from a cruciferous vegetable affecting both a signaling pathway and a catabolic process.

Processing blackcurrants dramatically reduces the content and does not enhance the urinary yield of anthocyanins in human subjects.

Blackcurrant (BC) fruits are a rich source of biologically active anthocyanins but little is known of the anthocyanin content of commonly consumed BC products or how processing affects the absorption. We report that whereas fresh and frozen whole BC were a rich source of anthocyanins, containing 897 and 642mg (100gFW)(-1) of total anthocyanins the levels in all other products were substantially lower (0.05-10.3% of the levels in fresh fruit). Further, when the absorption and excretion of BC was assessed in volunteers consuming a portion (100g) of frozen whole BC (642mg total anthocyanins) and, 300g of a BC drink made by diluting concentrated syrup (33.6mg total anthocyanins), only small quantities of BC anthocyanins were excreted in urine (fruit, 0.053±0.022%; drink, 0.036±0.043%; mean percent urinary yield±SD) and they were not detected in plasma. These data indicate that fresh and frozen BC, but not processed products, are rich sources of anthocyanins but, regardless of the food source, these anthocyanins are poorly bioavailable.

Primary and secondary metabolite composition of kernels from three cultivars of Portuguese chestnut (Castanea sativa Mill.) at different stages of industrial transformation.

Chestnut (Castanea sativa) is an important basic food in rural diets and a major starch crop used in a similar way to potatoes. Chestnuts are a fundamental economic resource in the "chestnut regions" not only for the fruit but also for the chestnut wood. Chestnuts have become increasingly important with respect to human health, for example, as an alternative gluten-free flour source. Chestnuts are also a rich source of other beneficial compounds, but there have been few studies on the composition during processing. In this study, we analyzed the chemical composition of three Portuguese cultivars at different stages of industrial processing. The chestnut cultivars were Longal, Judia, and Martaínha. All three cultivars had high moisture contents but were low in ash, crude fat, and crude protein contents, with high starch and low fiber contents. The free amino acid contents, including various essential amino acids, varied depending on the cultivar. All three cultivars also had a significant content of polyphenolics with gallic acid; ellagic acid was predominant among hydrolyzable and condensed tannins. Many of these compounds are known to exert significant positive effects on human health. The one-way analysis of variance for fresh chestnut shows significant differences among the three cultivars for most of the studied parameters. The same statistical analysis applied to each one of the two cultivars (Judia and Longal) sampled for the four processing steps analyzed indicates a significant effect of this factor in practically all of the constituents. On the other hand, the two-way analysis of variance shows that, besides the residual, the processing step and the interaction cultivar x processing step were the factors that more contributed for the total variation observed in the constituents analyzed, while the contribution of cultivar was much less significant.

Identification and quantification of glucosinolates in sprouts derived from seeds of wild Eruca sativa L. (salad rocket) and Diplotaxis tenuifolia L. (wild rocket) from diverse geographical locations.

The Brassicaceae rocket species Eruca sativa L. (salad rocket) and Diplotaxis tenuifolia L. (wild rocket) are consumed throughout the world in salads, predominantly the leaves but also the flowers and more recently the sprouts (seedlings). Ontogenic profiling of glucosinolates and flavonoids in plants derived from commercial seed of these species has previously been done, but no studies have been conducted to determine how geographical origin affects glucosinolate composition in rocket species. Seeds from wild E. sativa L. and D. tenuifolia L. from diverse regions of the world were obtained from gene banks and grown under controlled conditions. Sprouts were harvested when they would normally be harvested for consumption, and glucosinolates were extracted and profiled in these accessions. All of the sprouts from Italian E. sativa L. had consistently high total glucosinolate content, with only a few exceptions, and also the highest percentage contents of 4-mercaptobutylglucosinolate. In contrast, sprouts produced from Central and Eastern European seeds had a much higher percentage of 4-methylthiobutylglucosinolate. With a single exception, Tunisia, all sprouts produced from North African seeds had very high 4-methylthiobutylglucosinolate contents. The single sample from China had a high total glucosinolate content and glucosinolate profile that was very similar to the accessions from Uzbekistan and Pakistan. All of the D. tenuifolia L. sprouts had consistently high total glucosinolate contents, and a high percentage of this was 4-mercaptobutylglucosinolate. This glucosinolate variation in levels and profiles of the rockets can be used for genetic studies, selected breeding, and human intervention studies.

Enzymatic hydrolysis of flavonoids and pectic oligosaccharides from bergamot (Citrus bergamia Risso) peel.

Pectinolytic and cellulolytic enzymes (Pectinase 62L, Pectinase 690L, and Cellulase CO13P) were used to evaluate the solubilization of carbohydrates and low molecular weight flavonoids from bergamot peel, a major byproduct of the essential oil industry. The enzymes were characterized for main-chain and side-chain polysaccharide hydrolyzing activities and also against pure samples of various flavonoids previously identified in bergamot peel to determine various glycosidase activities. The addition of Pectinase 62L or 690L alone, or the combination of Pectinase 62L and Cellulase CO13P, was capable of solubilizing between 70 and 80% of the bergamot peel, and up to 90% of the flavonoid glycosides present were cleaved to their aglycones. Cellulase CO13P alone solubilized 62% of the peel but had no deglycosylating effect on the flavonoid glycosides. Over a 24-h time course, a rapid release of cell wall carbohydrates was observed after treatment with Pectinase 62L, with a concurrent gradual hydrolysis of the flavonoid glycosides. Size-exclusion chromatography of the solubilized extract showed that after 24-h incubation, the majority of the solubilized carbohydrates were present as monosaccharides with a smaller proportion of oligosaccharides.

Assessment of the anti-genotoxic, anti-proliferative, and anti-metastatic potential of crude watercress extract in human colon cancer cells.

Although it is known to be a rich source of the putative anti-cancer chemicals isothiocyanates, watercress has not been extensively studied for its cancer preventing properties. The aim of this study was to investigate the potential chemoprotective effects of crude watercress extract toward three important stages in the carcinogenic process, namely initiation, proliferation, and metastasis (invasion) using established in vitro models. HT29 cells were used to investigate the protective effects of the extract on DNA damage and the cell cycle. The extract was not genotoxic but inhibited DNA damage induced by two of the three genotoxins used, namely hydrogen peroxide and fecal water, indicating the potential to inhibit initiation. It also caused an accumulation of cells in the S phase of the cell cycle indicating (possible) cell cycle delay at this stage. The extract was shown to significantly inhibit invasion of HT115 cells through matrigel. Component analysis was also carried out in an attempt to determine the major phytochemicals present in both watercress leaves and the crude extract. In conclusion, the watercress extract proved to be significantly protective against the three stages of the carcinogenesis process investigated.

Ontogenic profiling of glucosinolates, flavonoids, and other secondary metabolites in Eruca sativa (salad rocket), Diplotaxis erucoides (wall rocket), Diplotaxis tenuifolia (wild rocket), and Bunias orientalis (Turkish rocket).

As an influence of the Mediterranean diet, rocket species such as Eruca sativa L., Diplotaxis species, and Bunias orientalis L. are eaten all over the world at different ontogenic stages in salads and soups. They are all species within the plant order Capparales (glucosinolate-containing species), and all are from the family Brassicaceae. Predominantly, the leaves of these species are eaten raw or cooked, although Eruca flowers are also consumed. There is considerable potential with raw plant material for a higher exposure to bioactive phytochemicals such as glucosinolates, their hydrolysis products, and also phenolics, flavonoids, and vitamins such as vitamin C. These compounds are susceptible to ontogenic variation, and the few published studies that have addressed this topic have been inconsistent. Thus, an ontogenic study was performed and all samples were analyzed using a previously developed robust liquid chromatography/mass spectrometry method for the identification and quantification of the major phytochemicals in all tissues of the rocket species. Seeds and roots of both Eruca and Diplotaxis contained predominantly 4-methylthiobutylglucosinolate. Leaves of Eruca and Diplotaxis contained high amounts of 4-mercaptobutylglucosinolate with lower levels of 4-methylthiobutlyglucosinolate and 4-methylsulfinylbutylglucosinolate. Flowers of Eruca and Diplotaxiscontained predominantly 4-methylsulfinylbutyl-glucosinolate. In addition, roots of both Diplotaxisspecies contained 4-hydroxybenzylglucosinolate but 4-hydroxybenzylglucosinolate was absent from roots of Eruca. Seeds and seedlings of all Eruca contained N-heterocyclic compounds but no sinapine, whereas Diplotaxis contained sinapine but not the N-heterocycles. In all tissues of B. orientalis, 4-hydroxybenzylglucosinolate and 4-methylsulfinyl-3-butenylglucosinolate were predominant. All rocket tissues, except roots, contained significant levels of polyglycosylated flavonoids, with/without hydroxycinnamoyl acylation. The core aglycones were kaempferol, quercetin, and isorhamnetin. The exception was B. orientalis, which had a negligible seed flavonoid content as compared with the other species. Anthocyanins were only detected in Eruca flowers and consisted of a complex pattern of at least 16 different anthocyanins.