Cardiotonic glycosides are extracted mostly from leaves of Digitalis plants. Commercial production of bioactive secondary metabolites by traditional agriculture is an inefficient process and can be affected by climatic and soil conditions. Strategies, based on in vitro culture methods, have been extensively studied to improve the production of specific plant derived chemicals. The aim of the present research was to obtain biomass of D. purpurea using the temporary immersion system (TIS) and to determine the content of cardiotonic glycosides (digitoxin, digoxin and lanatoside C) as secondary metabolites of commercial value for the pharmaceutical industry. Shoots were cultured in 1,000 ml TIS during 28 days. The effect of four immersion frequencies (once every 2, 4, 6, and 12 h) was studied. Biomass accumulation was influenced by immersion frequency. The maximum biomass accumulation (values in respect of fresh and dry weight) was obtained with immersions every 4 h (six immersions per day). HPLC analysis revealed the presence of digoxin and digitoxin for all immersion frequencies. No lanatoside C was detected in the biomass cultured in TIS. Digoxin concentrations varied depending on the frequencies tested. In contrast, the digitoxin content showed no dependency on the immersion frequency. Net production of digoxin and digitoxin per TIS were found to be higher with immersions every 4 h. The best net production of digitoxin and digoxin per TIS were 167.6 and 119.9 lg, respectively. The development of organ culture based on temporary immersion system can be a reliable method for the steady production of biomass for cardiotonic glycosides production, which is reported for the first time for Digitalis genus in this communication.

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The biomass production of Cymbopogon citratus shoots cultivated in bioreactors according to the temporary immersion (TIS) principle was assessed under different growth conditions. The effect of gassing with CO 2 -enriched air, reduced immersion frequency, vessel size and culture time on total phenolic and flavonoid content and free radical scavenging effect of the methanolic extracts was measured. From the TIS-culture of C. citratus, seven compounds were isolated and identified as caffeic acid (1), chlorogenic acid (2), neochlorogenic acid (3), p-hydroxybenzoic acid (4), p-hydroxybenzoic acid 3-O--d-glucoside (5), glutamic acid (6) and luteolin 6-C-fucopyranoside (7). The occurrence of compounds 1Ð7 and their variability in C. citratus grown under different TIS conditions was determined by HPLC. The free radical scavenging effect of the methanolic extract and compounds was measured by the discoloration of the free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH). The main metabolites in 6-and 8-week-old cultures, both in 5 and 10 l vessels, were chlorogenic acid (2) (100Ð113 mg%) and neochlorogenic acid (3) (80Ð 119 mg%), while in the cultures with CO 2 -enriched air and reduced immersion frequency the main compound detected in the extracts was glutamic acid (6) (400 and 670 mg% for the green and white biomass and 619 and 630 mg% for the green and white biomass, respectively). The most active compounds, as free radical scavengers, in the DPPH discoloration assay were caffeic acid (1), chlorogenic acid (2), neochlorogenic acid (3) and the flavonoid luteolin 6-C-fucopyranoside (7).

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A rapid in vitro propagation system leading to the formation of shoots, calli, roots, cell suspensions and plantlets was developed for the Andean medicinal plant Fabiana imbricata (Solanaceae). Massive propagation of shoots and roots was achieved by the temporary immersion system (TIS), morphogenesis and maintenance of cell suspensions by standard in vitro culture techniques. Oleanolic acid (OA), rutin, chlorogenic acid (CA) and scopoletin content in aerial parts of wild growing Fabiana imbricata plants as well as in plantlets regenerated in vitro, callus cultures, cell suspensions and biomass, obtained by the TIS system was assessed by HPLC. On a dry weight basis, the OA content in the aerial parts of the plant ranged between 2.26 and 3.47% while in vitro plantlets, callus and root cultures presented values ranging from not detected up to 0.14%. The rutin content of the samples presented a similar trend with maxima between 0.99 and 3.35% for the aerial parts of the plants to 0.02 to 0.20% for plantlets, 0.12% for cell suspensions and 0.28% for callus. Rutin was not detected in the roots grown by the TIS principle. The CA and scopoletin content in the aerial parts of F. imbricata ranged between 0.22Ð1.15 and < 0.01Ð0.55%, respectively. In the plantlets, the concentration of CA was 0.29 to 1.48% with scopoletin in the range 0.09 to 0.64% while in the callus sample, the CA and scopoletin content were 0.46 and 0.66%, respectively. A very different result was found in roots grown by TIS, where both OA and rutin were not detected and its main secondary metabolite, scopoletin was found between a range of 0.99 and 1.41% with CA between of 0.11 and 0.42%.