Hydrogen peroxide-induced antioxidant activities and cardiotonic glycoside accumulation in callus cultures of endemic Digitalis species

• Exposure of H2O2 resulted in the highest level of cardiac glycosides.
• The predominant cardiac glycoside was lanatoside C.
• Oxidative stress increases activities of CAT and SOD in cultures.
• Treatment of callus with H2O2 drastically increased phenolic and proline content.

The effect of hydrogen peroxide (H2O2) on callus cultures of four Digitalis species (Digitalis lamarckii, Digitalis trojana, Digitalis davisiana and Digitalis cariensis) increased catalase (CAT), superoxide dismutase (SOD), total phenolic, proline activity and cardiotonic glycoside production. Callus derived from hypocotyl explants was cultured on Murashige and Skoog medium supplemented with 0.25 mg L−1 indole-3-acetic acid (IAA) and 0.5 mg L−1 thidiazuron (TDZ). After a month of culture, callus was transferred to MS medium containing 10 mM H2O2 and then incubated for 6 h. The amount of five cardenolides (Lanatoside C, Digitoxin, Digoxigenin, Gitoxigenin and Digoxin) as well as CAT, SOD, total phenolic, proline activity from Digitalis species were compared. No digoxin was detected in all treatments and control groups. The total cardenolides estimated were in the order of D. lamarckii (586.65  μg g−1 dw), D. davisiana (506.79 μg g−1 dw), D. cariensis (376.60 μg g−1 dw) and D. trojana (282.39 μg g−1 dw). It was clear that H2O2 pre-treatment resulted in an increase in enzymatic and nonenzymatic antioxidants. However, a significant negative relationship between cardenolides production and overall activities of CAT, SOD, total phenolic and proline was evident. The described protocol here will be useful for the development of new strategies for a large-scale production of cardenolides.