Evonik (France)

BackgroundThapsigargin and nortrilobolide are sesquiterpene lactones found in the Mediterranean plant Thapsia garganica L. Thapsigargin is a potent inhibitor of the sarco/endoplasmic reticulum calcium ATPase pump, inducing apoptosis in mammalian cells. This mechanism has been used to develop a thapsigargin-based cancer drug first by GenSpera and later Inspyr Therapeutics (Westlake Village, California). However, a stable production of thapsigargin is not established.ResultsIn vitro regeneration from leaf explants, shoot multiplication and rooting of T. garganica was obtained along with the production of thapsigargins in temporary immersion bioreactors (TIBs). Thapsigargin production was enhanced using reduced nutrient supply in combination with methyl jasmonate elicitation treatments. Shoots grown in vitro were able to produce 0.34% and 2.1% dry weight of thapsigargin and nortrilobolide, respectively, while leaves and stems of wild T. garganica plants contain only between 0.1 and 0.5% of thapsigargin and below detectable levels of nortrilobolide. In addition, a real-time reverse transcription PCR (qRT-PCR) study was performed to study the regulatory role of the biosynthetic genes HMG-CoA reductase (HMGR), farnesyl diphosphate synthase (FPPS), epikunzeaol synthase (TgTPS2) and the cytochrome P450 (TgCYP76AE2) of stem, leaf and callus tissues. Nadi staining showed that the thapsigargins are located in secretory ducts within these tissues.ConclusionsShoot regeneration, rooting and biomass growth from leaf explants of T. garganica were achieved, together with a high yield in vitro production of thapsigargin in TIBs.

Lilium candidum, also known as White lily, is a monocot that belongs to the Liliaceae family and naturally grows throughout Mediterranean countries and western Asia. Bulbs and flowers are the traditionally used organs for therapeutic purposes [1]. However, leaves and roots can also be interesting sources of natural compounds for the industry. The use of biotechnology and in vitro culture for the propagation of L. candidum provide a sustainable production, constant over the year, and free from any contaminants. Depending on the plant organ, secondary metabolites profile and compound yields change, making each tissue a possible source of different bioactive molecules. We are presenting the in vitro generation of Lilium candidum differentiated biomass, under solid medium and temporary immersion bioreactors (leaves and roots). Compounds were extracted and analysed by HPLC-DAD and UPLC-MS, enabling a direct comparison of the quantity of each metabolite depending on the source of the extract. Both differentiated tissues (leaves and roots) showed a significantly enriched extract with specific metabolic profile. The nature of bioactive compounds from leaves consisted of derivatives of jatropham (pyrroline alkaloid) and the main constituent was assigned to a glycosilated kaempferol (flavonoid). However, in root extracts, the jatropham derivatives were present in low amounts and many other peaks corresponding to phenylpropanoid glycerol glucosides (PGG) were highlighted by mass spectrometry. The differences in metabolite contents of both organs have a direct impact on the activities of each extract. Kaempferol as well as jatropham and their derivatives are related to a wide range of pharmacological activities like antioxidant, anti-inflammatory or even anticancer [2]. The in vitro propagation of L. candidum is a sustainable approach to obtain bioactive extracts which can provide specific activities useful for the pharmaceutical and cosmetic industries.