Chemical analysis of callus extracts from toxic and non-toxic varieties of Jatropha curcas L.

Jatropha curcas L. belongs to Euphorbiaceae family, and it synthesizes flavonoid and diterpene compounds that have showed antioxidant, anti-inflammatory, anticancer, antiviral, antimicrobial, antifungal and insecticide activity. Seeds of this plant accumulate phorbol esters, which are tigliane type diterpenes, reported as toxic and, depending on its concentration, toxic and non-toxic varieties has been identified. The aim of this work was to characterize the chemical profile of the extracts from seeds, leaves and callus of both varieties (toxic and non-toxic) of Jatropha curcas, to verify the presence of important compounds in dedifferentiated cells and consider the possibility of using these cultures for the massive production of metabolites. Callus induction was obtained using NAA (1.5 mg L-1) and BAP (1.5 mg L-1) after 21 d for both varieties. Thin layer chromatography analysis showed differences in compounds accumulation in callus from non-toxic variety throughout the time of culture, diterpenes showed an increase along the time, in contrast with flavonoids which decreased. Based on the results obtained through microQTOF-QII spectrometer it is suggested a higher accumulation of phorbol esters, derived from 12-deoxy-16-hydroxy-phorbol (m/z 365 [M+H]+), in callus of 38 d than those of 14 d culture, from both varieties. Unlike flavonoids accumulation, the MS chromatograms analysis allowed to suggest lower accumulation of flavonoids as the culture time progresses, in callus from both varieties. The presence of six glycosylated flavonoids is also suggested in leaf and callus extracts derived from both varieties (toxic and non-toxic), including: apigenin 6-C-α-L-arabinopyranosyl-8-C-ß -D-xylopyranoside (m/z 535 [M+H]+), apigenin 4'-O-rhamnoside (m/z 417 [M+H]+), vitexin (m/z 433 [M+H]+), vitexin 4'-O-glucoside-2″-O-rhamnoside (m/z 741 [M+H]+), vicenin-2 (m/z 595 [M+H]+), and vicenin-2,6″-O-glucoside (m/z 757 [M+H]+).

Improving the production of podophyllotoxin in hairy roots of Hyptis suaveolens induced from regenerated plantlets.

Ten Hyptis suaveolens hairy root lines were established by infecting nodal explants with K599+pGus-GFP+ and ATCC15834+pTDT strains from Agrobacterium rhizogenes. Genetic transformation was confirmed by epifluorescence and plagiotropic hairy root growth in absence of growth regulators. Cytotoxicity was determined using the sulforhodamine B method, and the production of podophyllotoxin (PTOX) was measured by high performance thin layer chromatography scanning. Through these methodologies, HsTD10 was identified as the hairy root line with the highest cytotoxicity and PTOX production, which was corroborated by liquid chromatography-mass spectrometry and micrOTOF-Q II. A suspension culture of HsTD10 was established in which PTOX and carbohydrate consumption during growth kinetics were quantified by high-performance liquid chromatography. Procedures to increase the production and retrieval of PTOX in the HsTD10 line included selection of culture medium, addition of thiamine, and modification of the PTOX extraction method. The best combination of these variables was MS medium at 75% of its components with the addition of 2 mg L-1 of thiamine, extraction with methanol-dichloromethane, and sonication at 40 ± 5°C. During kinetics, growth-associated PTOX accumulation was observed. The specific growth rate (µ) was 0.11 d-1. The highest concentration of PTOX obtained with HsTD10 (5.6 mg g-1 DW) was 100 times higher than that reported for roots of wild plants and 56 times higher than that for in vitro nontransformed roots of H. suaveolens.