Filamentous fungi from Plantago lanceolata L. leaves: Contribution to the pattern and stability of bioactive metabolites

Abstract

The aim of this study was to test contribution of plant-associated microorganism (PAMs) to metabolite stability/instability in a medicinal plant matrix.Therefore, PAM strains were isolated and identified based on relevant DNA sequences from Plantago lanceolata leaves. Sterile water extracts of P. lanceolata were incubated with the isolated strains and antioxidants (ascorbic acid (AA), and EDTA) for 15 days, and changes in the concentrations of chief bioactive constituents (aucubin, catalpol, acteoside (= verbascoside)) were quantified by capillary electrophoresis. Phenolic breakdown-products were identified by GC-MS.PAMs were identified from the genera Epicoccum, Bipolaris, Cladosporium, Leptosphaerulina, Aspergillus, Eurotium and Penicillium (pathongens, endophytes, and other species). Some fungi caused significant decomposition of the chief constituents (p < 0.001). Surprisingly, some strains inhibited breakdown of acteoside (p < 0.001). Meanwhile, concentration of several phenolic acids increased in fungi-infested extracts (p<0.001). Gentisic acid, 4-hydroxyphenyl acetic acid, 4-hydroxybenzoic acid and hydroxytyrosol were only present when the extract was infested with a PAM. The products are powerful antioxidants and chelators. Concentrations of phenolic acids influenced acteoside stability significantly (p < 0.01), as shown by basic data-mining techniques. AA and EDTA also significantly inhibited acteoside breakdown in sterile model solutions (p < 0.05).Our results suggest, that the phenolic acid mixture (produced during the fungal proliferation) protected acteoside from breakdown, possibly via its antioxidant activity and metal complexing ability.It was shown, that PAMs can increase or decrease the stability of chief metabolites in herbal matrices, and can significantly alter the chemical pattern of the plant matrix. 2 KeywordsPlantago lanceolata L., ribwort plantain, endophyte, plant-associated microorganism, metabolite stability, phenolic acids, acteoside 3

Effects of N source concentration and NH 4 + /NO 3 − ratio on phenylethanoid glycoside pattern in tissue cultures of Plantago lanceolata L.: A metabolomics driven full-factorial experiment with LC–ESI–MS 3

“…Higher concentrations of NH 4 + have led to decreased growth of the tissue cultures. This is in accordance with that described by Budzianowska et al (2004) and is usually attributed to the direct toxicity of excess NH 4 + at higher concentrations (George et al, 2007). Examination of the effects on growth was secondary, our main focus was to detect the changes in the metabolome, which will be detailed in the following sections.…”

“…This suggests that the availability of NO 3 − and NH 4 + in different ratios and net concentrations exerted more direct effects on biosynthesis of the examined NPs. These direct effects may include NH 4 + acidity stress (George et al, 2007), or other, unknown pathways.…”

“…Purification of acteoside and plantamajoside was described previously, the structures were confirmed by NMR Gonda et al, 2013). For all analytical applications, water purified with Arium 611 purification system (Sartorius) was used (conductivity 0.055 μS/cm).…”

“…Another significant problem is that intensive growth and intensive secondary metabolite biosynthesis rarely occurs simultaneously (Collin, 2001). The use of auxin hormones that enable the maintainability of TCs -especially 2,4-D -also silences lots of genes (George et al, 2007).…”

“…The N-source type and concentration, N/C-source ratio, C-source type and concentration (various mono-and disaccharides can be used), as well as potassium, phosphate, magnesium and many other components have effects on development and NP production in plant TCs. When optimizing a medium composition with respect to N source, the ratio of NH 4 + and NO 3 − , and the total amount of available N is usually optimized (George et al, 2007).…”