Derek Stewart

Phytochemicals of Brassicaceae in plant protection and human health – Influences of climate, environment and agronomic practice

In this review, we provide an overview of the role of glucosinolates and other phytochemical compounds present in the Brassicaceae in relation to plant protection and human health. Current knowledge of the factors that influence phytochemical content and profile in the Brassicaceae is also summarized and multi-factorial approaches are briefly discussed. Variation in agronomic conditions (plant species, cultivar, developmental stage, plant organ, plant competition, fertilization, pH), season, climatic factors, water availability, light (intensity, quality, duration) and CO(2) are known to significantly affect content and profile of phytochemicals. Phytochemicals such as the glucosinolates and leaf surface waxes play an important role in interactions with pests and pathogens. Factors that affect production of phytochemicals are important when designing plant protection strategies that exploit these compounds to minimize crop damage caused by plant pests and pathogens. Brassicaceous plants are consumed increasingly for possible health benefits, for example, glucosinolate-derived effects on degenerative diseases such as cancer, cardiovascular and neurodegenerative diseases. Thus, factors influencing phytochemical content and profile in the production of brassicaceous plants are worth considering both for plant and human health. Even though it is known that factors that influence phytochemical content and profile may interact, studies of plant compounds were, until recently, restricted by methods allowing only a reductionistic approach. It is now possible to design multi-factorial experiments that simulate their combined effects. This will provide important information to ecologists, plant breeders and agronomists.

Plant cell walls as dietary fibre : range, structure, processing and function

The ingestion of dietary fibre has been correlated with the prevention of many health-threatening diseases and cancers. Plant cell walls are the major source of dietary fibre and this review investigates the relationship between the structure of different types of plant cell walls and their beneficial effects. The effects of processing and cooking on dietary fibre are also examined. Structure-function relationships between individual cell wall components and the beneficial effects of dietary fibre are not well defined and it may be that the physical, physiochemical and topochemical properties of plant cell walls and their components are also important.

Berry extracts exert different antiproliferative effects against cervical and colon cancer cells grown in vitro.

Polyphenol-rich berry extracts were screened for their antiproliferative effectiveness using human cervical cancer (HeLa) cells grown in microtiter plates. Rowan berry, raspberry, lingonberry, cloudberry, arctic bramble, and strawberry extracts were effective but blueberry, sea buckthorn, and pomegranate extracts were considerably less effective. The most effective extracts (strawberry > arctic bramble > cloudberry > lingonberry) gave EC 50 values in the range of 25-40 microg/(mL of phenols). These extracts were also effective against human colon cancer (CaCo-2) cells, which were generally more sensitive at low concentrations but conversely less sensitive at higher concentrations. The strawberry, cloudberry, arctic bramble, and the raspberry extracts share common polyphenol constituents, especially the ellagitannins, which have been shown to be effective antiproliferative agents. However, the components underlying the effectiveness of the lingonberry extracts are not known. The lingonberry extracts were fractionated into anthocyanin-rich and tannin-rich fractions by chromatography on Sephadex LH-20. The anthocyanin-rich fraction was considerably less effective than the original extract, whereas the antiproliferative activity was retained in the tannin-rich fraction. The polyphenolic composition of the lingonberry extract was assessed by liquid chromatography-mass spectrometry and was similar to previous reports. The tannin-rich fraction was almost entirely composed of procyanidins of linkage type A and B. Therefore, the antiproliferative activity of lingonberry was caused predominantly by procyanidins.

Fourier-transform infrared and Raman spectroscopic evidence for the incorporation of cinnamaldehydes into the lignin of transgenic tobacco (Nicotiana tabacum L.) plants with reduced expression of cinnamyl alcohol dehydrogenase.

Xylem from stems of genetically manipulated tobacco plants which had had cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) activity down-regulated to a greater or lesser degree (clones 37 and 49, respectively) by the insertion of antisense CAD cDNA had similar, or slightly higher, lignin contents than xylem from wild-type plants. Fourier-transform infrared (FT-IR) microspectroscopy indicated that down-regulation of CAD had resulted in the incorporation of moieties with conjugated carbonyl groups into lignin and that the overall extent of cross-linking, particularly of guaiacyl (4-hydroxy-3-methoxyphenyl) rings, in the lignin had altered. The FT-Raman spectra of manipulated xylem exhibited maxima consistent with the presence of elevated levels of aldehydic groups conjugated to a carbon-carbon double bond and a guaiacyl ring. These maxima were particularly intense in the spectra of xylem from clone 37, the xylem of which exhibits a uniform red coloration, and their absolute frequencies matched those of coniferaldehyde. Furthermore, xylem from clone 37 was found to have a higher content of carbonyl groups than that of clone 49 or the wild-type (clone 37: clone 49: wild-type; 2.4:1.6:1.0) as measured by a degradative chemical method. This is the first report of the combined use of FT-IR and FT-Raman spectroscopies to study lignin structure in situ. These analyses provide strong evidence for the incorporation of cinnamaldehyde groups into the lignin of transgenic plants with down-regulated CAD expression. In addition, these non-destructive analyses also suggest that the plants transformed with antisense CAD, in particular clone 37, may contain lignin that is less condensed (cross-linked) than that of the wild-type.

Berry polyphenols inhibit α-amylase in vitro: identifying active components in rowanberry and raspberry

Polyphenol-rich extracts from a range of berries inhibited α-amylase in vitro, but the most effective were from raspberry and rowanberry (IC50 values of 21.0 and 4.5 μg/mL, respectively). The inhibitory components were examined by different approaches. Extracts from yellow and red raspberries were equally able to inhibit α-amylase. Because the yellow raspberry extracts effectively lacked anthocyanins, this suggested that they were not crucial for amylase inhibition. Notably, however, higher levels of other phenolic components in yellow raspberries (particularly, ellagitannins) did not increase amylase inhibition. Amylase inhibition in rowanberry was recovered in a fraction enriched in proanthocyanidins (PACs). Inhibition was ameliorated by bovine serum albumin, suggesting that PACs acted by binding to amylase. Co-incubation of rowanberry PACs with acarbose reduced the concentration of acarbose required for effective amylase inhibition. Such synergistic interactions could have implications for the current clinical use of acarbose for postprandial glycaemic control in type-2 diabetics.

Characterization and biosynthesis of non-degradable polymers in plant cuticles

Abstract. The structure and monomeric composition of the highly aliphatic and non-saponifiable fraction of cutans isolated from the leaf cuticles of Agave americana L. and Clivia miniata Reg. have been elucidated. Spectroscopic Fourier transform infrared and 13C-nuclear magnetic resonance, calorimetric and X-ray diffraction studies, together with biopolymer analysis after exhaustive ozonolysis, showed that the cutan fraction consists of an amorphous three-dimensional network linked by ether bonds containing double bonds and free carboxylic acid functions. Data obtained from fatty acid sorption indicated that the new biopolymer investigated here has a highly hydrophobic character constituting an additional barrier biopolymer in those cuticles where it is present. Labelled [14C]linoleic acid was preferentially incorporated into the non-ester part of C. miniata leaf disks in comparison with the cutin fraction of the cuticular membrane. This indicates that the cis-pentadiene system of polyunsaturated fatty acids is involved in the formation of intramolecular linkages, mainly ether bonds, of the aliphatic biopolymer.

Berry components inhibit α-glucosidase in vitro: Synergies between acarbose and polyphenols from black currant and rowanberry

Polyphenol-rich extracts from certain berries inhibited α-glucosidase activity in vitro. The two most effective berry extracts, from black currant and rowanberry, inhibited α-glucosidase with IC(50) values respectively of 20 and 30μg GAE/ml and were as effective as the pharmaceutical inhibitor, acarbose. These berry extracts differed greatly in their polyphenol composition: black currant was dominated by anthocyanins (∼70% of total) whereas rowanberry was enriched in chlorogenic acids (65% total) and had low levels of anthocyanins. Both black currant and rowanberry extracts potentiated the inhibition caused by acarbose and could replace the inhibition lost by reducing the acarbose dose. However, no additive effects were noted when black currant and rowanberry extracts were added in combination. The mechanisms underlying the synergy between acarbose and the berry polyphenols and the lack of synergy between the berry components are discussed. These extracts exhibited the potential to replace acarbose (or reduce the dose required) in its current clinical use in improving post-prandial glycaemic control in type 2 diabetics. As a result, these polyphenols may offer a dietary means for type 2 diabetics to exercise glycaemic control.

Towards fruitful metabolomics: high throughput analyses of polyphenol composition in berries using direct infusion mass spectrometry.

Tannin-enriched extracts from raspberry, cloudberry and strawberry were analysed by liquid chromatography-mass spectrometric (LC-MS) techniques. The raspberry and cloudberry extracts contained a similar mixture of identifiable ellagitannin components and ellagic acid. However, the strawberry extract contained a complex mixture of ellagitannin and proanthocyanidin components that could not be adequately resolved to allow identification of individual peaks. Nevertheless, the negative ESI-MS spectra obtained by direct infusion mass spectrometric (DIMS) analysis described the diversity of these samples. For example, the predominance of signals associated with Lambertianin C in cloudberry and Sanguiin H6 in raspberry tannin extracts could be discerned and the diversity of signals from procyanidin and propelargonidin oligomers could be identified in the strawberry extract. The dose response for the main ellagitannin-derived signals in the raspberry tannin sample revealed a saturation effect probably due to ion suppression effects in the ion trap spectrometer. Nevertheless, DIMS spectra of whole berry extracts described qualitative differences in ellagitannin-derived peaks in raspberry, cloudberry and strawberry samples. In addition, positive mode DIMS spectra illustrated qualitative differences in the anthocyanin composition of berries of progeny from a raspberry breeding population that had been previously analysed by LC-MS. This suggests that DIMS could be applied to rapidly assess differences in polyphenol content, especially in large sample sets such as the progeny from breeding programmes.

Anthocyanin-enriched bilberry and blackcurrant extracts modulate amyloid precursor protein processing and alleviate behavioral abnormalities in the APP/PS1 mouse model of Alzheimer's disease

A growing body of epidemiological evidence suggests that fruit and vegetable juices containing various phenolic compounds can reduce the risk of Alzheimers disease (AD). As the altered amyloid precursor protein (APP) processing leading to increased β-amyloid (Aβ) production is a key pathogenic feature of AD, we elucidated the effects of different polyphenols on neuroprotection and APP processing under different in vitro stress conditions. The effects of these compounds were also investigated in transgenic AD mice (APdE9). Free radical toxicity and apoptosis were induced in human SH-SY5Y neuroblastoma cells overexpressing APP751. Menadione-induced production of reactive oxygen species was significantly decreased upon treatment with myricetin, quercetin or anthocyanin-rich extracts in a dose-dependent manner. However, these extracts did not affect caspase-3 activation, APP processing or Aβ levels upon staurosporine-induced apoptosis. APdE9 mice fed with anthocyanin-rich bilberry or blackcurrant extracts showed decreased APP C-terminal fragment levels in the cerebral cortex as compared to APdE9 mice on the control diet. Soluble Aβ40 and Aβ42 levels were significantly decreased in bilberry-fed mice as compared to blackcurrant-fed mice. Conversely, the ratio of insoluble Aβ42/40 was significantly decreased in blackcurrant-fed mice relative to bilberry-fed mice. Both berry diets alleviated the spatial working memory deficit of aged APdE9 mice as compared to mice on the control diet. There were no changes in the expression or phosphorylation status of tau in APdE9 mice with respect to diet. These data suggest that anthocyanin-rich bilberry and blackcurrant diets favorably modulate APP processing and alleviate behavioral abnormalities in a mouse model of AD.

Transcriptional and metabolic profiles of Lolium perenne L. genotypes in response to a PEG‐induced water stress

Metabolic profiling was carried out in the forage grass Lolium perenne L. (perennial ryegrass) to uncover mechanisms involved in the plants response to water stress. When leaf and root materials from two genotypes, with a contrasting water stress response, were analysed by GC-MS, a clear difference in the metabolic profiles of the leaf tissue under water stress was observed. Differences were principally due to a reduction in fatty acid levels in the more susceptible Cashel genotype and an increase in sugars and compatible solutes in the more tolerant PI 462336 genotype. Sugars with a significant increase included: raffinose, trehalose, glucose, fructose and maltose. Increasing the ability of perennial ryegrass to accumulate these sugars in response to a water deficit may lead to more tolerant varieties. The metabolomics approach was combined with a transcriptomics approach in the water stress tolerant genotype PI 462336, which has identified perennial ryegrass genes regulated under water stress.