HPLC-PDA-ESI-HRMS-Based Profiling of Secondary Metabolites of Rindera graeca Anatomical and Hairy Roots Treated with Drought and Cold Stress

Abstract

To cope with environmental harmful conditions, plant cells developed adaptive strategy that involves production of a wide variety of complex secondary metabolites. The spectrum and quantity of biosynthesized compounds in specific plant species is determined by its genotype, tissue, developmental and physiological stage and environmental factors. This phenomenon was used to exploit the potential of anatomical and hairy root cultures of Rindera graeca to produce bioactive compounds. Cultivated in vitro roots were subjected to abiotic stresses i.e., drought or coldness. Next the extract profiling was performed using HPLC-PDA-ESI-HRMS method, as well quantitative determination of caffeic, rosmarinic and lithospermic B acids, that were present in all root extracts. Phenolic acids, flavonoids and iridoids represent the major groups of compounds detected in chemical profiles growing under various conditions roots. The highest number of phytochemicals was determined in roots subjected to coldness. Lithospermic B acid proved to be the most abundant compound in all investigated extracts. Among applied abiotic stress factors it was demonstrated that coldness affected to the most secondary metabolites production. The results of current study suggest that root cultures of R. graeca could serve as a new and abundant source of lithospermic B acid.

Keywords: Boraginaceae rosmarinic acid; abiotic stresses; chemical profile; in vitro cultures; lithospermic B acid; total phenolic and total flavonoid content.

Figure 1

(a) Total phenolic (TPC) and (b) total flavonoid (TFC) content determined in R. graeca roots cultivated under various conditions. RgAR—anatomical roots; RgTR7—hairy root line TR7; RgTR17—hairy root line TR17; time “0”—28-day-old roots at time of inoculation; Control—roots cultivated without any treatment for 14 days; Drought stress—roots treated by drought stress for 14 days; Cold stress—roots treated by cold stress for 14 days. The same letters indicate statistically significant differences (p ≤ 0.05) in relation to control within the same root lines between treatments. Asterisks (*) indicate statistically significant differences (p ≤ 0.05) in relation to time “0” within the same root lines between treatments.

Figure 2

HPLC-PDA chromatograms (wavelength 320 nm) of RgAR line root extracts:(A) 28-day-old (time zero); (B) 14-day old untreated roots-control; (C) roots treated 14 days with drought stress; (D) roots treated 14 days with cold stress.

Figure 3

HPLC-PDA chromatograms (wavelength 320 nm) of RgTR7 line root extracts: (A) 28-day-old (time zero); (B) 14-day old untreated roots-control; (C) roots treated 14 days with drought stress; (D) roots treated 14 days with cold stress.

Figure 4

HPLC-PDA chromatograms (wavelength 320 nm) of RgTR17 line root extracts: (A) 28-day-old (time zero); (B) 14-day old untreated roots-control; (C) roots treated 14 days with drought stress; (D) roots treated 14 days with cold stress.