Natural diversity of hydroxycinnamic acid derivatives, flavonoid glycosides, carotenoids and chlorophylls in leaves of six different amaranth species.

Amaranth species are globally grown food crops. However, knowledge about the composition of their secondary metabolites is insufficient. Here, selected hydroxycinnamic acid derivatives, flavonoid glycosides, carotenoids and chlorophylls in the leaves of 14 genotypes from six different amaranth species were identified and quantified. For the first time, caffeic acid esters of isocitric and several aldaric acids were isolated and quantified in a leafy food matrix. High concentrations of hydroxycinnamic acid derivatives and chlorophylls, and moderate amounts of flavonoids and carotenoids were detected. A hierarchical clustering method of the metabolic profiles followed by Random Amplification of Polymorphic DNA (RAPD)-PCR fingerprinting was used to group the genotypes. Using this combined approach, three main groups of amaranth species were assigned. The information provided in this study increases the attractiveness of the amaranth genus as a food crop due to its strong diversity of plant secondary metabolites that are associated with numerous health-promoting benefits.

Evaluation of an Aqueous Extract from Horseradish Root (Armoracia rusticana Radix) against Lipopolysaccharide-Induced Cellular Inflammation Reaction.

Horseradish (Armoracia rusticana) is a perennial crop and its root is used in condiments. Traditionally, horseradish root is used to treat bacterial infections of the respiratory tract and urinary bladder. The antiphlogistic activity, determined in activated primary human peripheral blood mononuclear cells (PBMC), was evaluated for an aqueous extract and its subfractions, separated by HPLC. Compound analysis was done by UHPLC-QToF/MS and GC-MS. The aqueous extract concentration-dependently inhibited the anti-inflammatory response to lipopolysaccharide (LPS) in terms of TNF-α release at ≥37 µg/mL. Further, the cyclooxygenase as well as lipoxygenase pathway was blocked by the extract as demonstrated by inhibition of COX-2 protein expression and PGE2 synthesis at ≥4 µg/mL and leukotriene LTB4 release. Mechanistic studies revealed that inhibition of ERK1/2 and c-Jun activation preceded COX-2 suppression upon plant extract treatment in the presence of LPS. Chemical analysis identified target compounds with a medium polarity as relevant for the observed bioactivity. Importantly, allyl isothiocyanate, which is quite well known for its anti-inflammatory capacity and as the principal pungent constituent in horseradish roots, was not relevant for the observations. The results suggest that horseradish root exerts an antiphlogistic activity in human immune cells by regulation of the COX and LOX pathway via MAPK signalling.

Nasturtium (Indian cress, Tropaeolum majus nanum) dually blocks the COX and LOX pathway in primary human immune cells.

BACKGROUND: Nasturtium (Indian cress, Tropaeolum majus) is known for its pharmacological value in the treatment of bacterial infections of the upper air tract and urinary bladder. However, scientific data on the anti-inflammatory potency in human-derived cells is missing. PURPOSE: The aim of this study was to investigate the potential of nasturtium to inhibit the lipopolysaccharide (LPS) induced inflammatory response in primary human cells of the immune system. STUDY DESIGN: The anti-inflammatory activities of nasturtium and its fractions were evaluated via regulation of arachidonic acid (AA) pathway and MAPK kinase cascade. Fraction H4 which was responsible for the anti-inflammatory effects was further characterized. METHODS: Human peripheral blood mononuclear cells (PBMC) were either treated with plant extracts or fractions thereof, stimulated with LPS and/or N-formyl-methionyl-leucyl-phenylalanine (fMLP) and analysed for COX and LOX, release of prostaglandin PGE2, leukotriene LTB4, TNF-alpha and ERK signaling pathway activation. The plant extracts were separated into four fractions by HPLC; fraction H4 was subjected to UHPLC-ToF/MS analysis to identify potential bioactive compounds. RESULTS: We found that aqueous extracts of nasturtium did exert strong concentration dependent suppression of LPS-triggered TNF-alpha release and COX pathway signaling, including PGE2 synthesis. Whereas COX-1 protein expression was not impacted, LPS-triggered COX-2 protein expression was concentration dependently blocked by the plant extract but not COX-2 enzyme activity. These findings suggest a mechanism of action for the plant extract which is different from non-steroidal anti-inflammatory drugs (NSAIDs). Moreover, the plant extract blocked leukotriene LTB4 release, the major end product of the 5-LOX pathway from PBMC. Down-regulation of ERK1/2 and c-Jun activation preceded COX-2 suppression upon plant extract treatment in the presence of LPS. Using HPLC separation of the aqueous extract followed by metabolomic analysis we could limit the number of relevant bioactive compounds in the extract to about 50. CONCLUSIONS: This study provides a rationale for the anti-inflammatory efficacy of nasturtium observed in man and gives first insight into the underlying molecular mechanisms.

Absorption of red clover isoflavones in human subjects: results from a pilot study.

In addition to soya-derived preparations, red clover-based dietary supplements have gained considerable interest as an alternative isoflavone (IF) source. While metabolism and bioavailability of the main IF from both sources have already been investigated, studies are still lacking on the biokinetic behaviour of IF, which are present in red clover in minor amounts. In the present pilot study, in which seven volunteers ingested a single dose of a commercial red clover dietary supplement, we focused on the absorption of three such IF, irilone (IRI), prunetin (PRUN) and pseudobaptigenin (PBAP). The compounds were measured as aglycones after enzymatic hydrolysis. A single intake of an amount of as low as 3.8 mg IRI (out of 38.8 mg IF in total) resulted in an IRI plasma concentration of 0.35 (sd 0.16) mum at 6.5 h post-ingestion. Compared to the plasma concentrations found for daidzein (0.39 mum) and genistein (0.06 mum), expected to be the main IF metabolites in plasma, the present findings indicate that IRI might possess a relatively high bioavailability. Furthermore, PRUN and PBAP were detected in human plasma for the first time.

The red clover isoflavone irilone is largely resistant to degradation by the human gut microbiota.

Intestinal bacteria may influence bioavailability and physiological activity of dietary isoflavones. We therefore investigated the ability of human intestinal microbiota to convert irilone and genistein in vitro. In contrast to genistein, irilone was largely resistant to transformation by fecal slurries of ten human subjects. The fecal microbiota converted genistein to dihydrogenistein, 6'-hydroxy-O-desmethylangolensin, and 2-(4-hydroxyphenyl)-propionic acid. However, considerable interindividual differences in the rate of genistein degradation and the pattern of metabolites formed from genistein were observed. Only one metabolite, namely dihydroirilone, was formed from irilone in minor amounts. In further experiments, Eubacterium ramulus, a prevalent flavonoid-degrading species of the human gut, was tested for transformation of irilone. In contrast to genistein, irilone was not converted by E. ramulus. Irilone only differs from genistein by a methylenedioxy group attached to the A-ring of the isoflavone skeleton. This substitution obviously restricts the degradability of irilone by human intestinal bacteria.

Application of LC and GC hyphenated with mass spectrometry as tool for characterization of unknown derivatives of isoflavonoids.

Polyphenols belonging to the class of secondary metabolites of plants and microorganisms play an important role as bioactive food constituents as well as contaminants. Structure elucidation of polyphenols in plant extracts or polyphenol metabolites, especially those arising during biotransformation, still represents a challenge for analytical chemistry. Various approaches have been proposed to utilize fragmentation reactions in connection with mass spectrometry (MS) for structural considerations on polyphenolic targets. We compiled and applied specific liquid chromatography (LC)-electrospray ionization in positive mode [ESI(+)]-tandem MS (MS/MS) and gas chromatography (GC)-(electron impact, EI)-MS/MS fragmentation reactions with a special focus on the analysis of isoflavones, whereby this technique was also found to be extendable to determine further polyphenols. For ESI(+)-MS the basic retro-Diels-Alder (rDA) fragmentation offers information about the substitution pattern in the A- and B-rings of flavonoids and the elimination of a protonated 4-methylenecyclohexa-2,5-dienone (m/z = 107) fragment can be used as a diagnostic tool for many isoflavanones. For GC-(EI)-MS/MS analysis after derivatization of the analytes to their trimethylsilyl ethers, the elimination of methyl radicals, tetramethylsilane groups or the combined loss of two methyl groups can be shown to be specific for certain substitution patterns in polyphenols. The applicability of the fragmentation reactions presented is demonstrated exemplarily for three derivatives of the isoflavone irilone. With the help of these fragmentation reactions of the two MS techniques combined, a reliable identification of polyphenols is possible. Especially in such cases where NMR cannot be utilized owing to low analyte amounts being available or prior to purification, valuable information can be obtained.