A two-liquid-phase bioreactor was designed to extract indole alkaloids from Catharanthus roseus hairy roots with silicon oil. Partition studies between silicon oil and culture medium showed that the silicon oil did not alter the availability of nutrients. The affinity of tabersonine and löchnericine for silicon oil is nine times higher than for the aqueous phase. Cultures were elicited with 25 mg/L of jasmonic acid. The growth of the hairy roots was not significantly modified by the presence of silicon oil. The overall specific yields of tabersonine and löchnericine were increased by 100-400% and 14-200%, respectively, with the use of silicon oil in nonelicited control cultures. In elicited cultures, these values were 10-55% for tabersonine and 20-65% for löchnericine. Serpentine was never found in the silicon oil. All measured alkaloids' specific yields were higher using silicon oil and elicitation, suggesting that the silicon oil, while acting as a metabolic sink for tabersonine and löchnericine, was efficient in increasing metabolic fluxes of the secondary metabolism pathways.

The effect of contact between cells and extractive phase on secondary metabolite production was investigated in two-phase suspension cultures of Eschscholtzia californica. A system was designed to extract benzophenanthridine alkaloids from the cell culture, without contact between XAD-7 resins and the cells: only medium was recirculated through a column packed with the extractive phase. This strategy was compared to the classic method of addition of resins directly into the cell suspension. Removal of the product directly from the medium enabled important increases in production of alkaloids, namely a 20-fold increase in sanguinarine production and a 10-fold increase in chelerythrine, with high recovery in the resin. The recirculation strategy greatly simplified the production process since the resins are easily recovered from the cell culture and enable harvest of product without termination of culture. However, due to limited flow rate, the recirculation strategy was slightly less effective than direct addition of resins into the cell suspension. In addition to enabling increased production, removal of secondary metabolites from the medium changed metabolic flux distribution, testifying to a complex control mechanism of production.

A rapid and precise analytical HPLC method has been developed for screening the major benzophenanthridine alkaloids produced by cell cultures of Eschscholtzia califomica, namely, sanguinarine, chelirubine, macarpine, chelerythrine and chelilutine. Separation was achieved on a C18, reversed-phase column with gradient elution using acetonitrile and 50 mM phosphoric acid. Detection was performed by both fluorescence (lambda(ex) 330 nm, lambda(em) 570 nm) and photodiode array, leading to good selectivity and precision in determining peak purity. A simple and quick sample preparation protocol was elaborated involving a methanolic extraction for the measurement of intracellular concentrations of the alkaloids and a solid phase extraction for their quantification in culture medium. Owing to the non-availability of commercially standards, a method for the purification of chelirubine, macar pine and chelilutine by semi-preparative HPLC was developed. Coupled together, the isolation method and the analytical method were highly reliable for screening the alkaloids of interest produced by E. califomica.