Transport of flavonolignans to the culture medium of elicited cell suspensions of Silybum marianum

Cell suspension cultures of Silybum marianum are able to excrete silymarin compounds into the medium upon elicitation with methyl jasmonate or cyclodextrins. Knowledge of transport mechanism is important to understand Sm metabolism and to develop strategies aimed at increasing production by means of cell cultures. For these reasons, a pharmacological approach was undertaken in this work in order to elucidate the possible mechanism involved in the release of this class of secondary metabolites into the extracellular medium of suspensions. Treatment with an ionophore or NH4Cl displayed little effect in elicited cultures, thus indicating that secondary transport, which uses electrochemical gradients, is not involved in the release. Several inhibitors of ABC transporters showed differential effects. Sodium ortho-vanadate, a typical suppressor of ATPase activity, was highly toxic to cultures even at very low concentrations. The common Ca-channel blocker verapamil did not influence extracellular secondary metabolite accumulation. Glybenclamide and probenecid, both effective inhibitors of ABCC-type ABC transporters, strongly reduced silymarin secretion. A partial cDNA, SmABC1, which showed similarity to ABCC-type ABC transporters, was isolated by RT-PCR from silymarin-producing cultures. SmABC1 expression was enhanced by methyljasmonate and cyclodextrins. Brefeldin A, a fungal metabolite which affects vesicular trafficking by preventing GTP/GDP exchange, inhibited release in a dose dependent manner. These results suggest that excretion of silymarin and their precursors is a transporter-dependent active transport and that yet another mechanism involving a vesicle trafficking system seems to participate in driving this class of secondary metabolites to the extracellular compartment. (C) 2013 Elsevier GmbH. All rights reserved.