Gordon J. McDougall

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.

Bioactive compounds in berries relevant to human health

Berries contain powerful antioxidants, potential allergens, and other bioactive compounds. Genetic and environmental factors affect production and storage of such compounds. For this reason breeding and biotechnological approaches are currently used to control or to increase the content of specific health-related compounds in fruits. This work reviews the main bioactive compounds determining the nutritional quality of berries, the major factors affecting their content and activity, and the genetic options currently available to achieve new genotypes able to provide, under controlled cultivation conditions, berries with the proper balance of bioactive compounds for improving consumer health.

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.

Colon-available raspberry polyphenols exhibit anti-cancer effects on in vitro models of colon cancer

Background There is a probable association between consumption of fruit and vegetables and reduced risk of cancer, particularly cancer of the digestive tract. This anti-cancer activity has been attributed in part to anti-oxidants present in these foods. Raspberries in particular are a rich source of the anti-oxidant compounds, such as polyphenols, anthocyanins and ellagitannins. Methods A colon-available raspberry extract (CARE) was prepared that contained phytochemicals surviving a digestion procedure that mimicked the physiochemical conditions of the upper gastrointestinal tract. The polyphenolic-rich extract was assessed for anti-cancer properties in a series of in vitro systems that model important stages of colon carcinogenesis, initiation, promotion and invasion. Results The phytochemical composition of CARE was monitored using liquid chromatography mass spectrometry. The colon-available raspberry extract was reduced in anthocyanins and ellagitannins compared to the original raspberry juice but enriched in other polyphenols and polyphenol breakdown products that were more stable to gastrointestinal digestion. Initiation – CARE caused significant protective effects against DNA damage induced by hydrogen peroxide in HT29 colon cancer cells measured using single cell microgelelectrophoresis. Promotion – CARE significantly decreased the population of HT29 cells in the G1 phase of the cell cycle, effectively reducing the number of cells entering the cell cycle. However, CARE had no effect on epithelial integrity (barrier function) assessed by recording the trans-epithelial resistance (TER) of CACO-2 cell monolayers. Invasion – CARE caused significant inhibition of HT115 colon cancer cell invasion using the matrigel invasion assay. Conclusion The results indicate that raspberry phytochemicals likely to reach the colon are capable of inhibiting several important stages in colon carcinogenesis in vitro.

Mind the gap—deficits in our knowledge of aspects impacting the bioavailability of phytochemicals and their metabolites—a position paper focusing on carotenoids and polyphenols

Various secondary plant metabolites or phytochemicals, including polyphenols and carotenoids, have been associated with a variety of health benefits, such as reduced incidence of type 2 diabetes, cardiovascular diseases, and several types of cancer, most likely due to their involvement in ameliorating inflammation and oxidative stress. However, discrepancies exist between their putative effects when comparing observational and intervention studies, especially when using pure compounds. These discrepancies may in part be explained by differences in intake levels and their bioavailability. Prior to exerting their bioactivity, these compounds must be made bioavailable, and considerable differences may arise due to their matrix release, changes during digestion, uptake, metabolism, and biodistribution, even before considering dose‐ and host‐related factors. Though many insights have been gained on factors affecting secondary plant metabolite bioavailability, many gaps still exist in our knowledge. In this position paper, we highlight several major gaps in our understanding of phytochemical bioavailability, including effects of food processing, changes during digestion, involvement of cellular transporters in influx/efflux through the gastrointestinal epithelium, changes during colonic fermentation, and their phase I and phase II metabolism following absorption.

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.

Changes in cell wall-associated peroxidases during the lignification of flax fibres

Abstract Cell wall-associated peroxidases were extracted selectively from fibre-bearing tissue dissected from the stem of Linum usitatissimum and their activities correlated with the extent of fibre lignification. The onset of fibre lignification was accompanied by increases in the levels of the peroxidases both ionically and covalently bound to the wall. However, the rise in the levels of covalently bound wall peroxidases was more dramatic. A portion of the covalently bound peroxidases could be isolated for study by solubilization with the glycanase mixture, driselase, whilst others remained firmly attached to the wall residue. The ability of driselase-solubilized wall peroxidases to oxidize NADH to form hydrogen peroxide rose sharply as the fibre cells became progressively lignified. This suggests that this process is accompanied by an increase in peroxidases covalently bound to the wall that have an affinity for the generation of hydrogen peroxide. A survey of the peroxidase isozymes present in both fractions using non-denaturing polyacrylamide gel electrophoresis identified a number of cationic and anionic isozymes that may have specific roles in the lignification of flax fibres.

Isolation and partial characterisation of the non-cellulosic polysaccharides of flax fibre

Abstract The non-cellulosic polysaccharides of flax fibre cells were isolated using chemical extraction methods. Extraction of mature retted flax fibre with the calcium chelating agent trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid (CDTA) removed polysaccharides enriched in galacturonic acid. These pectic polysaccharides were fractionated by their solubility in water into two fractions that differed in their neutral sugar content. Extraction of the fibre with aqueous 24% (w/v) potassium hydroxide produced the first hemicellulosic fraction (Hc1), which was enriched in xylose and, to a lesser extent, glucose- and galactose-containing polysaccharides. Digestion of aliquots of this fraction using the fungal carbohydrase mixture, Driselase, yielded xylobiose and isoprimeverose, which are characteristic limit digestion products of xylans and xyloglucans, respectively. Digestion of Hc1 with a crude cellulase preparation from Aspergillus niger yielded oligosaccharide products, one of which was similar to a characteristic nonasaccharide subunit of xyloglucan. Further extraction of the fibre residue with aqueous 18% (w/v) potassium hydroxide supplemented with 4% (w/v) of boric acid removed a second hemicellulosic fraction (Hc2), which was enriched in mannose-containing polysaccharides. Digestion of this fraction with a highly purified endo-(1 → 4)-β- d -mannanase converted 67% of the total polysaccharide into oligosaccharides within one hour. The β- d -mannanase-derived oligosaccharides had a monosaccharide composition of mannose-glucose of 1.6:1.0. Analysis of the oligosaccharides by TLC and high-performance anion exchange chromatography revealed products characteristic of a glucomannan similar to those found in hardwoods. The presence of β-linked d -glucose residues was confirmed by the removal of glucose from the β- d -mannanase-derived oligosaccharides by a purified β- d -glucosidase. A final extraction of the fibre with aqueous 90% Me2SO containing 4% (w/v) of boric acid removed a further pectic fraction (Hc3) enriched in neutral sugars. Preliminary analyses suggest that this fraction may contain a polysaccharide akin to rhamnogalacturonan I.

Identification and partial characterization of a coniferyl alcohol oxidase from lignifying xylem of Sitka spruce (Picea sitchensis)

Abstract. Oxidase activity in the developing xylem of branches of Sitka spruce [Picea sitchensis] (Bong) Carr. was expressed in synchrony with the deposition of lignin. The activity was closely associated with the cell wall but it could be extracted by elution with salt solutions such as 1u2009Mu2009NaCl or CaCl2. A number of different oxidase isoforms with isoelectric points in the range 8–5 were present in these cell wall extracts. These enzymes displayed a marked preference for the oxidation of coniferyl alcohol and efficiently initiated polymerization of coniferyl alcohol into insoluble, lignin-like polymers. They also had a substrate preference and profile of sensitivity to inhibitors that was dissimilar to those reported for classical catechol oxidase or laccase-type polyphenol oxidases. A novel procedure that combines extraction and affinity chromatography on Concanavalin-A to select high-mannose-type glycoproteins provided oxidase activity at higher purity and yield than previously used methods. A single band of oxidase activity (apparent Mr approx. 84 kDa) which was capable of oxidizing α-naphthol/N,N,N′N′-tetramethyl p-phenylene diamine in the absence of added hydrogen peroxide was detected in these cell wall extracts using non-denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The addition of hydrogen peroxide did not intensify the staining of this band but it confirmed the presence of a true peroxidase band of apparent Mr approx. 40 kDa. The properties of this coniferyl alcohol oxidase are different from those of laccase-type polyphenol oxidases (EC 1.10.3.2) previously implicated in lignin deposition in tree species, and their possible roles in this process are discussed.

Cell-Wall-Associated Peroxidases and Lignification during Growth of Flax Fibres

Summary Cell-wall associated peroxidases were selectively extracted from two defined areas of the flax stem throughout the vegetative development of the plant. The activities of these peroxidases were correlated with the extent of fibre lignification in these stem areas. Lignification of fibres in the hypocotyl (below the cotyledonary node) was well advanced by 5 weeks after emergence but lignification of fibres in the first internode (above the cotyledonary node) was only just evident at this time. The extent of lignification increased at both sites over the next 10 weeks. Extracellular peroxidases and wall bound peroxidases from the hypocotyl and first internode samples were able to oxidise a number of different substrates. Total wall-associated activity reached a maximum level around 7 to 9 weeks which coincided with the period of greatest development of the xylem. Between 11 to 15 weeks there is still considerable wall-associated peroxidase activity present at a time of increasing lignification of fibre cells. Fibre-bearing tissue was dissected from the central xylem core. Enzyme extracts obtained from this tissue had high peroxidase activity and showed little contamination from intracellular constituents. Non-denaturing acidic and basic gel electrophoresis of extracellular samples revealed several anionic and cationic peroxidase isozymes. There were no major qualitative or quantitative differences in the isozyme patterns from plants of different ages. Therefore, it was not possible to assign peroxidases which were specifically involved in fibre lignification. A comparison of peroxidases derived from flax callus with those from stem samples revealed differences in anionic and cationic peroxidase isozymes.