Anne G. Mortensen

Flavonoids in roots of white clover: interaction of arbuscular mycorrhizal fungi and a pathogenic fungus

The effects of two arbuscular mycorrhizal fungi (AMF) (Glomus mosseae and G. claroideum) and a pathogenic fungus (Pythium ultimum) on the production of eight flavonoids in roots of two white clover (Trifolium repens L.) cultivars were evaluated. Quantification of AM and pathogenic fungi in the roots showed that the AM symbiosis significantly reduced P. ultimum biomass and in some cases prevented infection. The flavonoid productions in clover roots varied depending on the presence of beneficial and/or pathogenic fungi, fungal isolate or plant cultivar. Only plants colonized with G. claroideum showed detectable concentrations of either coumestrol or kaempferol (cultivar-dependant). In addition, inoculation with G. claroideum resulted in significantly higher concentrations of coumestrol in cv. Sonja and medicarpin in cv. Milo. A low production of coumestrol and kaempferol in mycorrhizal plants may be G. mosseae-specific. Only the concentrations of formononetin and daidzein increased in clover roots in response to infection with P. ultimum. These flavonoids are supposedly stress metabolites, synthesized or produced from glycosides in response to pathogen infection. However, the presence of one or both AMF significantly lowered the formononetin and daidzein concentrations, and overruled the inductive effect of P. ultimum. Therefore the antagonistic action of AM against the pathogen must take place through another mechanism.

Transformation kinetics of 6-methoxybenzoxazolin-2-one in soil

Wheat (Triticum aestivum L.) and other cereals produce allelochemicals as natural defense compounds against weeds, fungi, insects and soil-borne diseases. The main benzoxazinoid allelochemical of wheat is 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), bound as β-glucoside and released upon plant injury. When leached from wheat to soil, DIMBOA is microbially transformed to 6-methoxy-benzoxazolin-2-one (MBOA). Exploiting benzoxazinoids and their degradation products as substitutes for synthetic pesticides depends on knowledge of transformation pathways and kinetics. In an MBOA degradation experiment at a concentration of 2400 nmol g–1 soil, the previously identified transformation products 2-amino-7-methoxy-phenoxazin-3-one (AMPO) and 2-acetylamino-7-methoxy-phenoxazin-3-one (AAMPO) were quantified. Three different kinetic models were applied to MBOA transformation kinetics; single first-order (SFO), first-order multi-compartment, and double first-order in parallel. SFO proved to be adequate and was subsequently applied to the transformations of MBOA, AMPO and AAMPO. Degradation endpoints, expressed as degradation time (DT), were calculated for MBOA, AMPO and AAMPO to test whether the maximum values for synthetic pesticides set by the European Commission and the Danish Environmental Protection Agency were exceeded. DT50 values for MBOA and AMPO were 5.4 d and 321.5 d, respectively, and DT90 values were 18.1 d and 1068 d, respectively. The DT50 value for AMPO exceeded the maximum value. The persistence, concentrations and toxicity of metabolites such as AMPO should be considered when breeding cereal crops with increased levels of benzoxazinoids.

Synthesis and quantitation of six phenolic amides in Amaranthus spp.

Cinnamoylphenethylamines are phenolic amides in which cinnamic acid provides the acid moiety and phenethylamine the amine moiety. Single ion monitoring (SIM) in LC-MS was performed on amaranth leaf extracts. Masses corresponding to sets of regioisomers, including previously reported compounds, were examined. Six peaks were detected and their corresponding standards synthesized for a quantitative LC-MS/MS investigation of cinnamoylphenethylamines in amaranth. Four cinnamoylphenethylamines (caffeoyltyramine, feruloyldopamine, sinapoyltyramine, and p-coumaroyltyramine) are reported in the Amaranthaceae for the first time; also, one rare compound, feruloyl-4-O-methyldopamine, appeared to be quite common in the genus Amaranthus. Feruloyldopamine showed moderate antifungal activity toward an isolate of Fusarium culmorum. Our LC-MS approach, in conjunction with the straightforward synthesis, provides a simple, reliable way of quantitatively investigating cinnamoylphenethylamines in plants. Concentrations of cinnamoylphenethylamines vary widely: feruloyltyramine was present in quantities of 5.26 to 114.31 microg/g and feruloyldopamine in quantities of 0.16 to 10.27 microg/g, depending on the plant sample.

Plasma and urine concentrations of bioactive dietary benzoxazinoids and their glucuronidated conjugates in rats fed a rye bread-based diet.

Thorough knowledge of the absorption and metabolism of dietary benzoxazinoids is needed to understand their health-promoting effects. In this study, the fates of these bioactive compounds were examined by LC-MS/MS in plasma, urine, and feces after ingesting a daily dose of 4780 ± 68 nmol benzoxazinoids from rye bread using Wistar rats as a model. HBOA-glc (2-β-D-glucopyranosyloxy-1,4-benzoxazin-3-one) was the predominant benzoxazinoid in the plasma (74 ± 27 nmol/L), followed by DIBOA-glc (2-β-D-glucopyranosyloxy-4-hydroxy-1,4-benzoxazin-3-one) and HBOA. The total level of benzoxazinoids in the urine was 1176 ± 66 nmol/d, which corresponds to approximately 25% of the total dietary intake. The urinary benzoxazinoid profile differed from that of plasma with HBOA-glc and DIBOA-glc (647 ± 31 and 466 ± 33 nmol/d, respectively) as the major urinary components. The glucuronide conjugates of HBOA and DIBOA were detected in both the plasma and urine. N-dehydroxylation was found to be a critical step in the absorption of hydroxamic acids. This unprecedented study will trigger future interest in the biological effects of benzoxazinoids in whole grain rye and wheat diets in humans and other animals.

Variation of polyphenols and betaines in aerial parts of young, field-grown Amaranthus genotypes.

Amaranthus hybridus and Amaranthus mantegazzianus are commonly cultivated and the entire young fresh plants consumed as vegetables in regions of Africa and Asia. A. hybridus and A. mantegazzianus were cultivated at four sites in three climate regions of the world: Santa Rosa, Argentina; Lleida, Spain; and Prague and Olomouc, both in the Czech Republic. The contents of flavonoids (isoquercitrin, rutin, nicotiflorin), hydroxybenzoic acids (protocatechuic acid, 4-hydroxybenzoic acid, vanillic acid), hydroxycinnamic acids (caffeic acid, p-coumaric acid, ferulic acid), hydroxycinnamyl amides (N-trans-feruloyltyramine, N-trans-feruloyl-4-O-methyldopamine), and betaines (glycinebetaine, trigonelline) were determined. The variation in phytochemical content due to species and cultivation site was analyzed utilizing the multivariate statistical methods of principal component analysis (PCA) and graphical model (GM). The Argentinean samples differed from the three other locations due to higher contents of most compounds. The samples from Spain and the Czech Republic differed from each other in the content of the negatively correlated metabolites trigonelline and the flavonoids. The two amaranth species were separated primarily by a higher content of trigonelline and the two hydroxycinnamyl amides in A. mantegazzianus. The GM showed that the quantities of the different analytes within each compound group were intercorrelated except in the case of the betaines. The betaines carried no information on each other that was not given through correlations with other compounds. The hydroxycinnamic acids were a key group of compounds in this analysis as they separated the other groups from each other (i.e., carried information on all of the other groups). This study showed the contents of polyphenols and betaines in the aerial parts of vegetable amaranth to be very dependent on growth conditions, but also revealed that some of the compounds (trigonelline and the two hydroxycinnamyl amides) may be useful as features of a taxonomic classification.

Erratum to: Differences among five amaranth varieties (Amaranthus spp.) regarding secondary metabolites and foliar herbivory by chewing insects in the field

In this study, we determined the abundance of secondary metabolites present in leaves of five varieties of Amaranthus, described the community of chewing insects observed in the foliage and also quantified damage by folivore insects in the field. Three flavonoid glucosides (rutin, nicotiflorin and isoquercitin), nine phenolic compounds (coumaric, vanillic, caffeic, syringic, ferulic, sinapic, protocatechuic, salicylic and 4-hydroxybenzoic acid) and three betalains (amaranthine, iso-amaranthine and betanin) were found to be present in amaranth leaves. Flavonoids appeared in of all varieties analyzed, with rutin being the most important. Betalains occurred only in some varieties and at different proportions, and nine phenolic acids were observed in all the varieties, with the exception of sinapic acid. Significant differences in the chemical composition of the varieties were noted. A total of 17 species of chewing phytophagous insects were observed through visual counting in Amaranthus plants, with the order Coleoptera being the most important and having the highest diversity of species. The degree of herbivory differed significantly among the varieties. Multivariate regression analysis indicated that the eight analyzed compounds detected in the plants had significant linear relationships with herbivory in the field. However, to draw any conclusions relating the amount of any compound to the degree of herbivory damage is premature at this stage of the research.

Phytotoxic effect, uptake, and transformation of biochanin A in selected weed species.

Certain isoflavones are plant growth inhibitors, and biochanin A is a major isoflavone in clover species used for weed management. The effect of biochanin A on the monocot weed species Echinochloa crus-galli L. and Lolium perenne L. and dicot species Silene noctiflora L., Geranium molle L., and Amaranthus caudatus L. was evaluated in agar medium bioassays. S. noctiflora and G. molle root growth was progressively inhibited with increasing concentrations of biochanin A, whereas the monocot species were unaffected. With regard to the dicot species, S. noctiflora (EC(50) = 35.80 μM and EC(25) = 5.20 μM) was more susceptible than G. molle (EC(50), EC(25) > 400 μM). S. noctiflora, G. molle, and E. crus-galli root and shoot samples, representing a susceptible, a less susceptible, and a nonsusceptible species, respectively, were analyzed by LC-MS to quantify biochanin A and its transformation products. Biochanin A and its known transformation products genistein, dihydrobiochanin A, pratensein, and p-coumaric acid were quantified. Sissotrin was identified and quantified while assigning unknown peaks. The treated root samples contained more biochanin A, genistein, pratensein, and dihydrobiochanin A than shoot samples.