Metabolite profiling by means of GC-MS combined with principal component analyses of natural populations of Nectaroscordum siculum ssp. bulgaricum (Janka) Stearn.

Nectaroscordum siculum ssp. bulgaricum (Janka) Stearn (Allium siculum subsp. dioscoridis (Sm.) K. Richt.) is a traditional culinary spice from South-East Europe. Studies of N. siculum have focused mainly on the botanical and taxonomic characteristics of this species and there is no data available in the scientific literature about its metabolite profile. Thus, the aim of the current study was metabolite profiling of four wild populations of N. siculum grown in Bulgaria by gas chromatography coupled to mass spectrometry (GC-MS) and subsequent principal component analysis (PCA) of the data obtained. The identified primary metabolites (carbohydrates, amino acids, organic acids and lipids) are initial compounds for the biosynthesis of different plant secondary metabolites, such as polyphenols and flavour compounds with valuable biological activities for humans. The health benefits of the phenolic acids identified in this study have been a prerequisite for the implementation of N. siculum in different food systems in order to increase their quality and biological value.

Alkaloid profiles and acetylcholinesterase inhibitory activities of Fumaria species from Bulgaria.

GC-MS analysis of alkaloid profiles of five Fumaria species, naturally grown in Bulgaria (F. officinalis, F. thuretii, F. kralikii, F. rostellata and F. schrammii) and analysis of acetylcholinesterase inhibitory activity of alkaloid extracts were performed. Fourteen isoquinoline alkaloids were identified, with the principle ones being protopine, cryptopine, sinactine, parfumine, fumariline, fumarophycine, and fumaritine. Protopine contents, defined by HPLC analysis varied between 210.6 ± 8.8 µg/g DW (F. schrammii) and 334.5 ± 7.1 µg/g DW. (F. rostellata). While all of the investigated alkaloid extracts significantly inhibited acetylcholinesterase activity, the F. kralikii demonstrated the highest level of inhibition (IC(50) 0.13 ± 0.01 mg extract/mL).

Determination of triterpenic acids and screening for valuable secondary metabolites in Salvia sp. suspension cultures.

Plant in vitro cultures are a prospective alternative for biochemicals production, for example the triterpenes oleanolic and ursolic acid present in plants and cell cultures of Salvia sp. Our objective was to develop a suitable analysis protocol for evaluation of triterpenic acid yield in plant raw material and in vitro cultures supporting selection processes. Moreover, valuable bioactive compounds had to be revealed. Thus, different strategies enhancing the separation for a sensitive and effective HPLC-UV method were investigated and the developed method was validated for linearity, precision, accuracy, limits of detection and quantification. A baseline separation of these isomers enabled detection limits of below 0.4 microg/mL and quantification limits of about 1.2 microg/mL. Over the tested concentration range a good linearity was observed (R2 > 0.9999). The variations in the method were below 6% for intra- and inter-day assays of concentration. Recoveries were between 85-98% for both compounds using ethanol as extraction solvent. Additionally, metabolite profiling of cell suspension culture extracts by GC-MS has shown the production variability of different plant metabolites and especially the presence of plant phenols and sterols. These studies provide a method suitable for screening plant and cell culture productivity of triterpenic acids and highlighted interesting co-products of plant cell cultures.

Hairy root type plant in vitro systems as sources of bioactive substances.

"Hairy root" systems, obtained by transforming plant tissues with the "natural genetic engineer" Agrobacterium rhizogenes, have been known for more than three decades. To date, hairy root cultures have been obtained from more than 100 plant species, including several endangered medicinal plants, affording opportunities to produce important phytochemicals and proteins in eco-friendly conditions. Diverse strategies can be applied to improve the yields of desired metabolites and to produce recombinant proteins. Furthermore, recent advances in bioreactor design and construction allow hairy root-based technologies to be scaled up while maintaining their biosynthetic potential. This review highlights recent progress in the field and outlines future prospects for exploiting the potential utility of hairy root cultures as "chemical factories" for producing bioactive substances.

Optimization of rosmarinic acid production by Lavandula vera MM plant cell suspension in a laboratory bioreactor.

The all-round effect of dissolved oxygen concentration, agitation speed, and temperature on the rosmarinic acid production by Lavandula vera MM cell suspension was studied in a 3-L laboratory bioreactor by means of the modified Simplex method. Polynomial regression models were elaborated for description of the process of rosmarinic acid production (Y) in the bioreactor as a consequence of the variation of the dissolved oxygen (X(1)) concentration between 10% and 50%; agitation (X(2)) between 100 and 400 rpm; and temperature (X(3)) between 22 and 30 degrees C. The optimization made it possible to establish the optimal conditions for the biosynthesis of rosmarinic acid by L. vera MM: dissolved oxygen (X(1)*), 50% of air saturation; agitation (X(2)*), 400 rpm; and temperature (X(3)*), 29.9 degrees C, where maximal yield (Y(max)) of 3489.4 mg/L of rosmarinic acid was achieved (2 times higher compared with the shake-flasks cultivation).