High throughput screening of mutants of oat that are defective in triterpene synthesis.

The triterpenes are a large and diverse group of plant natural products that have important functions in plant protection and food quality, and a range of pharmaceutical and other applications. Like sterols, they are synthesised from mevalonate via the isoprenoid pathway, the two pathways diverging after 2,3-oxidosqualene. During triterpene synthesis 2,3-oxidosqualene is cyclised to one of a number of potential products, the most common of these being the pentacyclic triterpene beta-amyrin. Plants often produce complex mixtures of conjugated triterpene glycosides which may be derived from a single triterpene skeleton. The delineation, functional analysis and exploitation of triterpene pathways in plants therefore represent a substantial challenge. Here we have carried out high throughput screening to identify mutants of diploid oat (Avena strigosa) that are blocked in the early steps of triterpene synthesis. We also show that mutants that are affected in the first committed step in synthesis of beta-amyrin-derived triterpenes, and so are unable to cyclise 2,3-oxidosqualene to beta-amyrin (sad1 mutants), accumulate elevated levels of primary sterols. The major differences were in Delta-7-campesterol and Delta-7-avenasterol, which both increased several fold relative to wild-type levels. This is presumably due to accumulation of squalene and 2,3-oxidosqualene and consequent feedback into the sterol pathway, and is consistent with previous reports in which specific oxidosqualene cyclase inhibitors and elicitors of triterpene biosynthesis were shown to have inverse effects on the flux through the sterol and triterpene pathways.

L-alpha-glycerophosphocholine contributes to meat's enhancement of nonheme iron absorption.

In this research, our aim was to isolate and characterize the substance known as "meat factor," which is reported to enhance nonheme iron absorption. We used various analytical techniques, and the final step was a human study to measure the effect of a candidate compound on iron absorption. Lean beef was selected for study, as it is known to increase nonheme iron absorption. Cooked ground beef was homogenized and aliquots were taken through a simulated gastric and intestinal digestion. This was followed by purification using fast protein liquid chromatography. The fractions were collected and applied to a Caco-2 cell system designed to measure iron absorption using radioiron. Fractions with an enhancing effect were analyzed by mass spectrometry, nuclear magnetic resonance, and HPLC, and a proposed empirical formula was obtained for the substance in the most active fraction (C(8)H(20) NO(6)P). Tandem mass spectrometry was used to identify the compound as L-alpha-glycerophosphocholine (L-alpha) by comparing the spectra against authentic material. We added a commercially available food grade source of L-alpha to vegetarian lasagna, with and without 100 mg ascorbic acid (a known enhancer of nonheme iron absorption), at the same enhancer:iron molar ratio (2:1), and fed meals to 13 women of child-bearing age with low iron stores. The nonheme iron was labeled with stable isotopes of iron to provide a total dose per meal of 10 mg iron, and absorption was measured from erythrocyte incorporation. Nonheme iron absorption from lasagna was increased by the addition of either ascorbic acid (P = 0.010) or L-alpha (P = 0.023). We have identified L-alpha as a component of muscle tissue that enhances nonheme iron absorption, and this finding provides new opportunities for iron fortification of foods.

Metabolic footprinting as a tool for discriminating between brewing yeasts.

The characterization of industrial yeast strains by examining their metabolic footprints (exometabolomes) was investigated and compared to genome-based discriminatory methods. A group of nine industrial brewing yeasts was studied by comparing their metabolic footprints, genetic fingerprints and comparative genomic hybridization profiles. Metabolic footprinting was carried out by both direct injection mass spectrometry (DIMS) and gas chromatography time-of-flight mass spectrometry (GC-TOF-MS), with data analysed by principal components analysis (PCA) and canonical variates analysis (CVA). The genomic profiles of the nine yeasts were compared by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis, genetic fingerprinting using amplified fragment length polymorphism (AFLP) analysis and microarray comparative genome hybridizations (CGH). Metabolomic and genomic analysis comparison of the nine brewing yeasts identified metabolomics as a powerful tool in separating genotypically and phenotypically similar strains. For some strains discrimination not achieved genomically was observed metabolomically.

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.

Shall I compare thee to a GM potato?

A fundamental issue in the safety assessment of genetically modified crops is the question of whether unintentional changes have occurred in the crop plant as a consequence of the genetic modification. This question was addressed recently by using a powerful metabolite fingerprinting and metabolite profiling method to assess whether genetically modified potatoes are substantially similar to their corresponding conventional cultivars.

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.

Dihydrocaffeoyl polyamines (kukoamine and allies) in potato (Solanum tuberosum) tubers detected during metabolite profiling.

Four related phenolic amides previously undescribed from the species were revealed during metabolic profiling of potato (Solanum tuberosum) tubers. N(1),N(12)-Bis(dihydrocaffeoyl)spermine (kukoamine A) and N(1),N(8)-bis(dihydrocaffeoyl)spermidine were positively identified by comparison with authentic standards, while the structures N(1),N(4),N(12)-tris(dihydrocaffeoyl)spermine and N(1),N(4),N(8)-tris(dihydrocaffeoyl)spermidine are proposed for the other two metabolites. Each amide was present at several tens of micrograms per gram of dry matter. Several of these compounds were subsequently detected in other solanaceous species, such as tomato (Lycopersicon esculentum) and Nicotiana sylvestris. They appeared not to be present in Arabidopsis thaliana or Beta vulgaris. Bis(dihydrocaffeoyl)spermine isomers have previously been identified in only a single plant, the Chinese medicinal species Lycium chinense (Solanaceae), where they may account for some of the described biological activity. The other compounds have not until now been reported in vivo, though some of the equivalent hydroxycinnamoyl derivatives are known. The surprising discovery of kukoamine and allies in a range of solanaceous species including potato, a common food crop that has a long history of scientific investigation, provides exemplary evidence for the potential of the nontargeted techniques of metabolomics in studying plant metabolites.

Stability of the major allergen Brazil nut 2S albumin (Ber e 1) to physiologically relevant in vitro gastrointestinal digestion.

The major 2S albumin allergen from Brazil nuts, Ber e 1, was subjected to gastrointestinal digestion using a physiologically relevant in vitro model system either before or after heating (100 degrees C for 20 min). Whilst the albumin was cleaved into peptides, these were held together in a much larger structure even when digested by using a simulated phase 1 (gastric) followed by a phase 2 (duodenal) digestion system. Neither prior heating of Ber e 1 nor the presence of the physiological surfactant phosphatidylcholine affected the pattern of proteolysis. After 2 h of gastric digestion, approximately 25% of the allergen remained intact, approximately 50% corresponded to a large fragment of M(r) 6400, and the remainder comprised smaller peptides. During duodenal digestion, residual intact 2S albumin disappeared quickly, but a modified form of the 'large fragment' remained, even after 2 h of digestion, with a mass of approximately 5000 Da. The 'large fragment' comprised several smaller peptides that were identified, by using different MS techniques, as deriving from the large subunit. In particular, sequences corresponding to the hypervariable region (Q37-M47) and to another peptide (P42-P69), spanning the main immunoglobulin E epitope region of 2S albumin allergens, were found to be largely intact following phase 1 (gastric) digestion. They also contained previously identified putative T-cell epitopes. These findings indicate that the characteristic conserved skeleton of cysteine residues of 2S albumin family and, particularly, the intrachain disulphide bond pattern of the large subunit, play a critical role in holding the core protein structure together even after extensive proteolysis, and the resulting structures still contain potentially active B- and T-cell epitopes.

Profiling glucosinolates, flavonoids, alkaloids, and other secondary metabolites in tissues of Azima tetracantha L. (Salvadoraceae).

Azima tetracantha L. (needle bush; bee sting bush; Salvadoraceae) is used as a food and for various herbal medicines in Africa, India, and Madagascar, but there is very little information on the secondary metabolites in this species. High concentrations of N-methoxy-3-indolylmethyl-glucosinolate, a common glucosinolate of Brassica crops such as Brussels sprouts and broccoli, were found in the roots and seeds of A. tetracantha. Lower concentrations were detected in the stems and young leaves. The roots also contained another indole glucosinolate that was provisionally identified, from MS data and comparison with indole glucosinolate standards, as N-hydroxy-3-indolymethyl-glucosinolate. The roots, stems, and leaves contained neoascorbigen (the condensation product of N-methoxy-indole-3-carbinol and ascorbic acid). The seeds of A. tetracantha contained a complex mixture of 26 flavonoids predominantly as glycosides and acyl-glycosides, with traces of aglycones. The core aglycones of these flavonoids were identified as quercetin, isorhamnetin (3'-O-methylquercetin), rhamnetin (7-O-methylquercetin), and rhamnazin (7, 3'-di-O-methylquercetin). No flavonoids or anthocyanins were detected in other tissues, and procyanidins were undetectable. The dimeric piperidine alkaloids azimine, azcarpine, and carpaine were found in all tissues of A. tetracantha.

Isolation, identification and stability of acylated derivatives of apigenin 7-O-glucoside from chamomile (Chamomilla recutita [L.] Rauschert).

The major flavonoids in the white florets of chamomile (Chamomilla recutita [L.] Rauschert) were rapidly purified using a combination of polyamide solid-phase extraction and preparative HPLC. From the combined LC/MS, LC/MS/MS, and NMR data the apigenin glucosides were identified as apigenin 7-O-glucoside (Ap-7-Glc), Ap-7-(6"-malonyl-Glc), Ap-7-(6"-acetyl-Glc), Ap-7-(6"-caffeoyl-Glc), Ap-7-(4"-acetyl-Glc), Ap-7-(4"-acetyl,6"-malonyl-Glc), and a partially characterised apigenin-7-(mono-acetyl/mono-malonylglucoside) isomer. Malonyl and caffeoyl derivatives of Ap-7-Glc have not previously been identified in chamomile. The two mono-acetyl/mono-malonyl flavonoids have not previously been reported in any plant species. These acylated glucosides are unstable and degrade to form acetylated compounds or Ap-7-Glc. The degradation products formed are dependent on the extraction and storage conditions, i.e. temperature, pH and solvent.

Mass spectrometry and structural characterization of 2S albumin isoforms from Brazil nuts (Bertholletia excelsa).

Proteomic approaches have been used to characterise the main 2S albumin isoforms from Brazil nuts (Bertholletia excelsa). Whilst most isoforms ( approximately 10 discrete protein species) exhibited molecular masses of around 12 kDa with a high amino acid sequence homology, important charge heterogeneity was found, with pIs varying between 4.6 and 6.6, with one >or=7.0. Proteomic analysis showed that these corresponded to a total of six National Center for Biotechnology Information (NCBI) accessions and that three isoforms had been purified to homogeneity corresponding to gi/384327, 112754 and 99609. The latter sequence corresponds to an isoform, previously only identified at the nucleotide sequence level, had a slightly higher molecular weight (13.4 kDa), and with noticeable differences in the primary structure. Proteins corresponding to six different NCBI accessions were identified, the heterogeneity of which had been increased by posttranslational processing. Evidence was found of cyclization of the N-terminal glutamine residue in two isoforms, together with ragged C-termini, indicative of carboxypeptidase activity within the vacuole following posttranslational processing. No evidence of glycosylation was found. Circular dichroism (CD) and Fourier transform-infrared (FT-IR) spectroscopy indicated all the studied isoforms were predominantly alpha-helical in nature, but that the Mr 13400 species was structurally distinct, with a higher proportion of alpha-helical structure.

Screening crucifer seeds as sources of specific intact glucosinolates using ion-pair high-performance liquid chromatography negative ion electrospray mass spectrometry.

Seeds, of either commercial crucifer crops or some wild and weed relatives, were screened for intact glucosinolates using a previously developed ion-pair LC-MS method. This method, in contrast to GC-MS techniques, ensures the accurate measurement of all classes of glucosinolates. Many crucifer seeds contained very high concentrations of glucosinolates with low concentrations of additional pigments and secondary metabolites. The other common seed metabolites were cinnamoylcholine esters, for example, sinapine. Glucosinolates derived from homologues of l-methionine were characteristic of Brassica and related crucifer species. In addition, significant concentrations of 4-hydroxy-3-indolylmethylglucosinolate were found in the majority of Brassica species. Wild and weed species often had relatively simple glucosinolate profiles: either a single glucosinolate or a predominant glucosinolate together with trace amounts of others. Species identified with seed glucosinolate profiles suitable for purification included various Alyssum, Erysimum, and Iberis species for 3-methythiopropyl-glucosinolate and 3-methylsulfinylpropyl-glucosinolate and various Alyssum, Erysimum, and Lepidium species with very high concentrations of C4-C6 aliphatic glucosinolates. Seeds of Arabis, Barbarea, Lepidium, Moringa, and Sinapis species were good sources of aromatic glucosinolates, and Azima tetracantha was a good source for N-methoxy-3-indolylmethyl-glucosinolate. MS data are reported for all of the intact glucosinolates detected from the screening process.

Characterization of metabolites of hydroxycinnamates in the in vitro model of human small intestinal epithelium caco-2 cells.

Hydroxycinnamic acids are antioxidant phenolic compounds which are widespread in plant foods, contribute significantly to total polyphenol intakes, and are absorbed by humans. The extent of their putative health benefit in vivo depends largely on their bioavailability. However, the mechanisms of absorption and metabolism of these phenolic compounds have not been described. In this study, we used the in vitro Caco-2 model of human small intestinal epithelium to investigate the metabolism of the major dietary hydroxycinnamates (ferulate, sinapate, p-coumarate, and caffeate) and of diferulates. The appearance of metabolites in the medium versus time was monitored, and the various conjugates and derivatives produced were identified by HPLC-DAD, LC/MS, and enzyme treatment with beta-glucuronidase or sulfatase. Enterocyte-like differentiated Caco-2 cells have extra- and intracellular esterases able to de-esterify hydroxycinnamate and diferulate esters. In addition, intracellular UDP-glucuronosyltransferases and sulfotransferases existing in Caco-2 cells are able to form the sulfate and the glucuronide conjugates of methyl ferulate, methyl sinapate, methyl caffeate, and methyl p-coumarate. However, only the sulfate conjugates of the free acids, ferulic acid, sinapic acid, and p-coumaric acid, were detected after 24 h. The O-methylated derivatives, ferulic and isoferulic acid, were the only metabolites detected following incubation of Caco-2 cells with caffeic acid. These results show that the in vitro model system differentiated Caco-2 cells have the capacity to metabolize dietary hydroxycinnamates, including various phase I (de-esterification) and phase II (glucuronidation, sulfation, and O-methylation) reactions, and suggests that the human small intestinal epithelium plays a role in the metabolism and bioavailability of these phenolic compounds.

Absorption of hydroxycinnamates in humans after high-bran cereal consumption.

Hydroxycinnamic acids are a group of phenolic compounds that exhibit a wide range of in vitro chemoprotective and antioxidant properties. Cereals containing a high proportion of the bran layers are rich in ester-linked hydroxycinnamic acids, such as ferulic and diferulic acids. The present work investigated the absorption in humans of hydroxycinnamic acids from high-bran breakfast cereal (wheat). Plasma and urine samples from six volunteers were collected before and after cereal consumption and analyzed for total hydroxycinnamic acids content after beta-glucuronidase/sulfatase treatment both by HPLC-DAD and by LC-MS (SIM monitoring). High-bran cereal administration resulted in increased plasma ferulic and sinapic acid concentrations (maximum levels detected of approximately 200 and approximately 40 nM, respectively) with absorption peaks between 1 and 3 h. Increases of approximately 4-fold in ferulic acid and approximately 5-fold in feruloylglycine were detected in 24-h urine after consumption of the cereal. Most of the ferulic acid detected in urine and plasma was present as conjugates (feruloylglycine and/or glucuronides). Diferulic acids were undetectable. The data show that ferulic and sinapic acids are taken up in humans from dietary high bran wheat but that absorption is limited and may originate only from the free and soluble portions present in the cereal.

Profiling glucosinolates and phenolics in vegetative and reproductive tissues of the multi-purpose trees Moringa oleifera L. (horseradish tree) and Moringa stenopetala L.

Moringa species are important multi-purpose tropical crops, as human foods and for medicine and oil production. There has been no previous comprehensive analysis of the secondary metabolites in Moringa species. Tissues of M. oleifera from a wide variety of sources and M. stenopetala from a single source were analyzed for glucosinolates and phenolics (flavonoids, anthocyanins, proanthocyanidins, and cinnamates). M. oleifera and M. stenopetala seeds only contained 4-(alpha-l-rhamnopyranosyloxy)-benzylglucosinolate at high concentrations. Roots of M. oleifera and M. stenopetala had high concentrations of both 4-(alpha-l-rhamnopyranosyloxy)-benzylglucosinolate and benzyl glucosinolate. Leaves from both species contained 4-(alpha-l-rhamnopyranosyloxy)-benzylglucosinolate and three monoacetyl isomers of this glucosinolate. Only 4-(alpha-l-rhamnopyranosyloxy)-benzylglucosinolate was detected in M. oleifera bark tissue. M. oleifera leaves contained quercetin-3-O-glucoside and quercetin-3-O-(6' '-malonyl-glucoside), and lower amounts of kaempferol-3-O-glucoside and kaempferol-3-O-(6' '-malonyl-glucoside). M. oleifera leaves also contained 3-caffeoylquinic acid and 5-caffeoylquinic acid. Leaves of M. stenopetala contained quercetin 3-O-rhamnoglucoside (rutin) and 5-caffeoylquinic acid. Neither proanthocyanidins nor anthocyanins were detected in any of the tissues of either species.

Absorption/metabolism of sulforaphane and quercetin, and regulation of phase II enzymes, in human jejunum in vivo.

For the first time the human intestinal effective permeability, estimated from the luminal disappearance and intestinal metabolism of phytochemicals, sulforaphane and quercetin-3,4'-glucoside, as well as the simultaneous changes in gene expression in vivo in enterocytes, has been studied in the human jejunum in vivo (Loc-I-Gut). Both compounds as components of an onion and broccoli extract could readily permeate the enterocytes in the perfused jejunal segment. At the physiologically relevant, dietary concentration tested, the average effective jejunal permeability (Peff) and percentage absorbed (+/- S.D.) were 18.7 +/- 12.6 x 10-4 cm/s and 74 +/- 29% for sulforaphane and 8.9 +/- 7.1 x 10-4 cm/s and 60 +/- 31% for quercetin-3,4'-diglucoside, respectively. Furthermore, a proportion of each compound was conjugated and excreted back into the lumen as sulforaphane-glutathione and quercetin-3'-glucuronide. The capacity of the isolated segment to deconjugate quercetin from quercetin-3,4'-diglucoside during the perfusion was much higher than the beta-glucosidase activity of the preperfusion jejunal contents, indicating that the majority (79-100%) of the beta-glucosidase capacity derives from the enterocytes in situ. Simultaneously, we determined short-term changes in gene expression in exfoliated enterocytes, which showed 2.0 +/- 0.4-fold induction of glutathione transferase A1 (GSTA1) mRNA (p < 0.002) and 2.4 +/- 1.2-fold induction of UDP-glucuronosyl transferase 1A1 (UGT1A1) mRNA (p < 0.02). The changes in gene expression were also seen in differentiated Caco-2 cells, where sulforaphane was responsible for induction of GSTA1 and quercetin for induction of UGT1A1. These results show that food components have the potential to modify drug metabolism in the human enterocyte in vivo very rapidly.

Involvement of dehydroalanine and dehydrobutyrine in the addition of glutathione to nisin.

Nisin variants and fragments were reacted with glutathione, and the products of the reactions were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and liquid chromatography/mass spectrometry (LC-MS). Reactions between glutathione and either [Ala5]nisin or [Ala33]nisin resulted in products with two glutathione molecules conjugated to one nisin variant molecule. Only one glutathione molecule was added to [Ala5,Ala33]nisin. Fragmentation of the nisin molecule resulted in nisin 1-12, nisin 1-20, and nisin 1-32 fragments. Each fragment retained two dehydro residues, which subsequently underwent reaction with glutathione. The data indicated that the dehydroalanine residues of nisin are sites of addition for glutathione. Such addition renders the nisin molecule inactive.

Characterization and content of flavonoid glycosides in genetically modified tomato (Lycopersicon esculentum) fruits.

There is a growing interest in producing food plants with increased amounts of flavonoids because of their potential health benefits. Tomatoes contain small amounts of flavonoids, most of which are located in the peel of the fruit. It has been shown that flavonoid accumulation in tomato flesh, and hence an overall increase in flavonoid levels in tomato fruit, can be achieved by means of simultaneous overexpression of the maize transcription factors LC and C1. Fruit from progeny of two modified lines (2027 and 2059) was selected for a detailed analysis and individual identification of flavonoids, at different stages of maturity. Nine major flavonoids were detected in the flesh of transgenic ripe tomatoes. LC/NMR, LC/MS, and LC/MS/MS enabled us to identify these as kaempferol-3,7-di-O-glucoside (1), kaempferol-3-O-rutinoside-7-O-glucoside (2), two dihydrokaempferol-O-hexosides (3 and 4), rutin (5), kaempferol-3-O-rutinoside (6), kaempferol-3-O-glucoside (7), naringenin-7-O-glucoside (8) and naringenin chalcone (9), which were quantified by HPLC/DAD. All but 5, 6, and 9 were detected in tomato for the first time. The total flavonoid glycoside content of ripe transgenic tomatoes of line 2059 was about 10-fold higher than that of the controls, and kaempferol glycosides accounted for 60% of this. Kaempferol glycosides comprised around 5% of the flavonoid glycoside content of ripe control tomatoes (the rest was rutin and naringenin chalcone). The rutin concentration in both transgenic and control fruits was similar.

Metabolism of quercetin-7- and quercetin-3-glucuronides by an in vitro hepatic model: the role of human beta-glucuronidase, sulfotransferase, catechol-O-methyltransferase and multi-resistant protein 2 (MRP2) in flavonoid metabolism.

Quercetin-3- and quercetin-7-glucuronides are major products of small intestine epithelial cell metabolism (J. Nutr. 130 (2000) 2765) but it is not known if quercetin glucuronides can be further processed in the liver or if they are excreted directly. Using the HepG2 hepatic cell model, we show that highly purified quercetin-7- and quercetin-3-glucuronides can follow two pathways of metabolism: (i) methylation of the catechol functional group of both quercetin glucuronides (44% of quercetin-7-glucuronide at a rate of 2.6 nmol/hr/10(6) cells, and 32% of quercetin-3-glucuronide at a rate of 1.9 nmol/hr/10(6) cells, over 48 hr) or (ii) hydrolysis of the glucuronide by endogenous beta-glucuronidase followed by sulfation to quercetin-3'-sulfate (7% of quercetin-7-glucuronide at a rate of 0.42 nmol/hr/10(6) cells and 10% of quercetin-3-glucuronide at a rate of 0.61 nmol/hr/10(6) cells, over 48 hr). In contrast, quercetin-4'-glucuronide was not metabolised, and interestingly this is not a major product of the small intestine absorption process. The conversion of the quercetin-7- and quercetin-3-glucuronide to the mono-sulfate conjugate shows intracellular deglucuronidation by beta-glucuronidase activity, allowing transient contact of the free aglycone with the cellular environment. Inhibition of methylation using a catechol-O-methyltransferase inhibitor shifted metabolism towards sulfation, as indicated by an increase in quercetin-3'-sulfate formation (increase in rate to 1.13 and 1.43 nmol/hr/10(6) cells for quercetin-7-glucuronide and quercetin-3-glucuronide, respectively). Efflux of quercetin metabolites from HepG2 cells (methylated glucuronide and sulfate conjugates) was not altered by verapamil, a p-glycoprotein inhibitor, but efflux was competitively inhibited by MK-571, a multidrug resistant protein inhibitor, indicating a role for multidrug resistant protein in the efflux of quercetin conjugates from HepG2 cells. These results show that HepG2 cells can absorb and turnover quercetin glucuronides and that human endogenous beta-glucuronidase activity could modulate the intracellular biological activities of dietary antioxidant flavonoids.

Identification of the major glucosinolate (4-mercaptobutyl glucosinolate) in leaves of Eruca sativa L. (salad rocket).

The major and structurally unique glucosinolate (GLS) in leaves of Eruca sativa L. (salad rocket) was identified as 4-mercaptobutyl GLS. Both 4-methylthiobutyl GLS and 4-methylsulfinylbutyl GLS were also present, but at lower concentrations. The 4-mercaptobutyl GLS was observed to oxidise under common GLS extraction conditions, generating a disulfide GLS that may be reduced efficiently by tris(2-carboxyethyl) phosphine hydrochloride (TCEP) to reform the parent molecule. The identities of 4-mercaptobutyl GLS and of the corresponding dimeric GLS were confirmed by LC/MS, MS/MS and NMR. Myrosinase treatment of an enriched GLS fraction or of the purified dimer GLS generated a mixture of unique bi-functional disulfides, including bis-(4-isothiocyanatobutyl) disulfide (previously identified elsewhere). TCEP reduction of the purified dimer, followed by myrosinase treatment, yielded only 4-mercaptobutyl ITC. GLS-derived volatiles generated by autolysis of fresh seedlings and true leaves were 4-mercaptobutyl ITC (from the newly identified GLS), 4-methylthiobutyl ITC (from 4-methylthiobutyl GLS) and 4-methylsulfinylbutyl ITC (from 4-methylsulfinyl-butyl GLS); no unusual bi-functional disulfides were found in fresh leaf autolysate. These results led to the conclusion that, in planta, the new GLS must be present as 4-mercaptobutyl GLS and not as the disulfide found after extraction and sample concentration. This new GLS and its isothiocyanate are likely to contribute to the unique odour and flavour of E. sativa.