The aim of this work was to determine the effect of stress conditions caused by different light sources, i.e., blue LED (λ = 430 nm), red LED (λ = 670 nm), blue and red LED (70%:30%) and white LED (430–670 nm) on the growth and morphology of cultivated in vitro Dracocephalum forrestii shoot culture. It also examines the effects on bioactive phenolic compound production and photosynthetic pigment content, as well as on antioxidant enzyme activity (CAT, SOD, POD) and antioxidant properties. The most beneficial proliferation effect was observed under white LEDs (7.1 ± 2.1 shoots per explant). The white and blue lights stimulated the highest fresh weight gain, while red light induced the highest dry weight gain. The total phenolic acid content ranged from 13.824 ± 1.181 to 20.018 ± 801 mg g DW⁻¹ depending on light conditions. The highest content of rosmarinic acid was found in the control shoots (cultivated under fluorescent lamps), followed by culture grown under red light. All LED treatments, especially red and blue, increased salvianolic acid B content, and blue increased apigenin p-coumarylrhamnoside biosynthesis. The greatest ferric reduction activity was observed in shoots cultivated under red light, followed by blue; this is associated with the presence of the highest total phenol content, especially phenolic acids. Similarly, the highest DPPH radical scavenging potential was observed under red light followed by blue. This study proves that LEDs have emerged as significant support for directed in vitro propagation, taking advantage of specific stress responses on various light spectra. This study also showed how stress induced by different LED light spectra increases in Dracocephalum forrestii the synthesis of pharmacologically-active compounds. Hence, light stress may turn out to be a simpler alternative to metabolic engineering for improving the production of secondary metabolites of therapeutic value.

Salvia viridis is a rich source of pharmacologically-important polyphenolic compounds with anti-inflammatory, antioxidant and anticancer activities. The aim of the present study was to optimise the large-scale cultivation of sage shoots in the Plantform bioreactor. It evaluated the effect of culture duration (two or three weeks), the use of a semi-continuous system and the immersion frequency in a liquid medium (every 80 or 170 min) on the growth and accumulation of secondary metabolites. The content of bioactive phenolic acids and phenylethanoids in the obtained material was determined using HPLC. The most efficient growth parameters, expressed as fresh (26.99 g) and dry weight (3.51 g) as well as proliferation ratio (18.6), were observed for shoots grown in a fed-batch system, immersed every 80 min; this approach yielded a 33-fold biomass increase within four weeks. However, the most efficient production of bioactive compounds was achieved for shoots grown for three weeks in a batch system, immersed every 80 min: total phenolic acid, total phenylethanoid and total phenol contents of 18.3, 11.4 and 29.7 mg/g DW. In these conditions, within three weeks, total phenolic acid level was almost 10 times higher than that found in the aerial parts of four-month-old soil-grown plants with a similar phenylethanoid level. Hence, the described S. viridis shoot culture cultivated in the Plantform bioreactor may be an alternative efficient source of plant material rich in valuable compounds.

The reactions of radicals with human serum albumin (HSA) under reductive stress conditions were studied using pulse radiolysis and photochemical methods. It was proved that irradiation of HSA solutions under reductive stress conditions results in the formation of stable protein aggregates. HSA aggregates induced by ionizing radiation are characterized by unique emission, different from the UV emission of non-irradiated solutions. The comparison of transient absorption spectra and the reactivity of hydrated electrons ($e_{aq}^{-}$) with amino acids or HSA suggests that electron attachment to disulfide bonds is responsible for the transient spectrum recorded in the case of albumin solutions. The reactions of $e_{aq}^{-}$ and ${CO}_2^{•-}$ with HSA lead to the formation of the same products. Recombination of sulfur-centered radicals plays a crucial role in the generation of HSA nanoparticles, which are stabilized by intermolecular disulfide bonds. The process of creating disulfide bridges under the influence of ionizing radiation is a promising method for the synthesis of biocompatible protein nanostructures for medical applications. Our Raman spectroscopy studies indicate strong modification of disulfide bonds and confirm the aggregation of albumins as well. Low-temperature measurements indicate the possibility of electron tunneling through the HSA protein structure to specific CyS-SCy bridges. The current study showed that the efficiency of HSA aggregation depends on two main factors: dose rate (number of pulses per unit time in the case of pulse radiolysis) and the temperature of the irradiated solution.

Triacontanol (TRIA) has been reported to influence signal transduction in the crosstalk triggered by various stress factors. As a signal player, it is also known to affect many physiological processes, including enhancing the biosynthesis of secondary metabolites. Such knowledge can be used to direct or boost the production of bioactive secondary compounds without stress induction. Therefore, the aim of this study is to evaluate the use of TRIA as a factor stimulating the growth and production of bioactive compounds in the shoot culture of Dracocephalum forrestii. TRIA was applied at three concentrations (2.5, 5, and 10 µM), alone or in combination with phytohormones (6-benzylaminopurine and indole-3-acetic acid). After five weeks, growth and physiochemical parameters (chlorophyll content, antioxidant enzyme activity, and phenolic acid level) were determined. The results indicate that TRIA application significantly increased shoot dry weight, chlorophyll content, antioxidant enzyme activities (superoxide dismutase, peroxidase, and catalase), and total polyphenol level; it also influenced the multiplication ratio in combination with growth regulators. The greatest antioxidant enzyme activity was observed for 5 µM TRIA in hormone-free medium, while the most significant secondary metabolite production was obtained for phytohormone-containing medium supplemented with 10 µM TRIA: total phenolic acid content (19.4 mg/g dry weight) was twice that of the control. Hence, the TRIA application appears to be a valuable biotechnology technique for modifying plant metabolite production.

Dracocephalum forrestii is a medicinal plant growing in China. The aim of the present study was to large-scale cultivation of D. forrestii transformed shoots in a temporary immersion system based on previously-optimized Murashige and Skoog (MS) medium supplemented with 0.5 mg/L N-benzyl-9-(2-tetrahydropyranyl)-adenine (BPA) and 0.2 mg/L indole-3-acetic acid (IAA) and physical (under blue LED) conditions. Shoot proliferation, and biomass and secondary metabolite accumulation in the shoots were assessed after a three-week growth period in a RITA® bioreactor. The levels of polyphenols in four types of extract (hydromethanolic extracts – mixtures with a 20%, 50%, and 80% methanol content and infusion) were determined using high-performance liquid chromatography (HPLC). Within three weeks, the culture increased its biomass 283-fold, with a proliferation ratio of 40.5 shoots or/and buds per explants. The most efficient solvent for extraction of phenolic compounds from raw material turned out to be 80% methanol solution; the highest polyphenol content was 40 mg/g DW (dry weight) with acacetin rhamnosyl-trihexoside (12.97 mg/g DW) and rosmarinic acid (10.68 mg/g DW) predominating. The intensive growth of the biomass of the culture allowed 570 mg of polyphenolic compounds to be obtained per liter of the medium. The antioxidant potential of extract of D. forrestii shoots was evaluated using three free radical-scavenging tests, and the inhibition of lipid peroxidation assay. In the study, the cytotoxic, antibacterial and antifungal potentials of the extract were also determined.