Studies on the Accumulation of Secondary Metabolites and Evaluation of Biological Activity of In Vitro Cultures of Ruta montana L. in Temporary Immersion Bioreactors

Abstract

The present work focuses on in vitro cultures of Ruta montana L. in temporary immersion Plantform^TM bioreactors. The main aim of the study was to evaluate the effects of cultivation time (5 and 6 weeks) and different concentrations (0.1-1.0 mg/L) of plant growth and development regulators (NAA and BAP) on the increase in biomass and the accumulation of secondary metabolites. Consequently, the antioxidant, antibacterial, and antibiofilm potentials of methanol extracts obtained from the in vitro-cultured biomass of R. montana were evaluated. High-performance liquid chromatography analysis was performed to characterize furanocoumarins, furoquinoline alkaloids, phenolic acids, and catechins. The major secondary metabolites in R. montana cultures were coumarins (maximum total content of 1824.3 mg/100 g DM), and the dominant compounds among them were xanthotoxin and bergapten. The maximum content of alkaloids was 561.7 mg/100 g DM. Concerning the antioxidant activity, the extract obtained from the biomass grown on the 0.1/0.1 LS medium variant, with an IC₅₀ 0.90 ± 0.03 mg/mL, showed the best chelating ability among the extracts, while the 0.1/0.1 and 0.5/1.0 LS media variants showed the best antibacterial (MIC range 125-500 µg/mL) and antibiofilm activity against resistant Staphylococcus aureus strains.

Keywords: Ruta montana; alkaloids; antibacterial activity; antibiofilm formation; antioxidant activity; coumarins; in vitro cultures; phenolic compounds.

Figure 1

Ruta montana bioreactor culture (LS NAA/BAP 0.1/0.1 mg/L, 5-week growth cycle).

Figure 2

Free radical scavenging activity (DPPH assay) (A), reducing power (B), and ferrous ion chelating activity (C) of methanol extracts obtained from biomass of R. montana bioreactor cultures 2 grown on LS medium variant supplemented with different concentrations of NAA/BAP mg/L 293 (0.5/0.5, 0.1/0.5, 1.0/1.0, 0.1/0.1), after 5-week growth cycle. Reference standard: BHT (A,B), EDTA (C). Values are expressed as the mean ± SD (n = 3). Statistically significant differences between different variant are indicated as **** p < 0.0001.

Figure 3

The effect of methanol extracts obtained from the biomass of R. montana bioreactor cultures grown on LS medium variant supplemented with different concentrations of NAA/BAP mg/L (0.1/0.1, 0.1/0.5, 0.5/0.5, 0.5/1.0, 1.0/1.0), 5-week growth cycle, on S. aureus strains biofilm formation reduction. The reduction percentage of biofilm formation was calculated using the following formula: [(OD₄₉₂ nm with extract/OD ₄₉₂ nm without extract) × 100]. Statistically significant differences are indicated as * p < 0.05 vs. each control group.