Elicitation improves rosmarinic acid content and antioxidant activity in Thymus lotocephalus shoot cultures

Thymus lotocephalus G. Lopez & R. Morales is an aromatic species endemic to the south of Portugal with medicinal properties. The aim of this work was to evaluate the effect of elicitors, cytokinin, and different sucrose concentrations on chemical composition and antioxidant activity of extracts from in vitro regenerated shoots. Elicitors (YE: yeast extract, salicylic acid, and AgNO3) were added directly to the medium. Phytochemical analysis include evaluation of total phenolic (TPC), flavonoid (TFC), and rosmarinic acid (RAC) contents. The TPCs and TFCs were determined by Folin-Ciocalteu and aluminum chloride calorimetric assays, respectively. Rosmarinic acid (RA), the main compound found in the studied extracts, was quantified by liquid chromatography-photodiode array detector. Results showed that the cytokinin benzyladenine has an inhibitory effect on TPC, TFC and RAC, as well as on antioxidant activity. TPC was only significantly improved by the presence of YE (from 95.49 +/- 3.34 to 118.34 +/- 6.24 mg(GAE)/g(extract)) and TFCs were not affected by elicitation. The increase in sucrose concentration, from 2 to 4%, and the three elicitors tested, particularly YE, enhanced RACs (from 48.61 +/- 0.09 in the control medium to 78.57 +/- 0.99 mg/g(extract) in YE-elicited medium). In accordance with these results, the antioxidant activity measured by 2,2-diphenyl-1-picrylhydrazyl, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), and ferric reducing antioxidant power assays was significantly greater in extracts from elicited shoots. Overall, the results obtained suggest that shoots of T. lotocephalus are a good source of antioxidant compounds and showed that the production of RA can be promoted by altering in vitro culture conditions.