Enhancing the production of scopoletin and quercetin 3-O-β-d-glucoside from cell suspension cultures of Tilia americana var. mexicana by modulating the copper and nitrate concentrations

Abstract

The aerial tissues of Tilia americana var. mexicana produce compounds with anxiolytic activity, such as quercetin 3-O-β-d-glucoside and tiliroside, in addition to ones with anti-inflammatory properties, such as scopoletin. These three compounds were initially identified in callus cultures of apical buds. In the present study, suspension cultures from leaf explant callus were established; the accumulation of scopoletin and quercetin 3-O-β-d-glucoside in these cultures were found to be cell-growth-associated using cell growth and active compound-production kinetics assays. The effects of varying the nitrate and copper concentrations in Murashige and Skoog (MS, 27.4 mM total nitrates and 0.01 µM copper) medium on the growth of a suspension culture of T. americana cells and on the production of active compounds were tested by means of central composite design (CCD) generally used in the response surface methodology (RSM). Cell growth, measured as maximal biomass, improved when the total nitrate concentration decreased in the MS medium to 13.7 mM (p < 0.01) regardless of the copper concentration. As a phytoalexin, scopoletin accumulated rapidly in plants after pathogen infection, in the suspension cultures scopoletin yield was stimulated by increased copper concentration to 1.2 μM (p < 0.01). According to the C:N hypothesis, the carbon excess generated by nitrates reduced to 8.03 mM (p < 0.01) stimulated the production of quercetin 3-O-β-d-glucoside. Cell suspension of T. americana represents a potential biotechnological alternative for industrial exploitation in a stirred-tank bioreactor using a two-phase process: (1) the first step will be to grow the cell suspension, (2) the second stage will consist in handle the suspension culture towards the production of anxiolytic compounds or towards the production of anti-inflammatory compounds. As well as to evaluate another elicitors to stimulate tiliroside production in the T. americana suspension cultures.