Mladenka Ilieva

Biosynthesis and radical scavenging activity of betalains during the cultivation of red beet (Beta vulgaris) hairy root cultures.

Betalains biosynthesis and antiradical scavenging activity were investigated during cultivation of four hairy root cultures of Beta vulgaris, obtained from different cultivars (Bordo, Egyptian, Detroit 2 and Detroit Dark Red). The best producer of betalains was a hairy root culture from Beta vulgaris cv. Detroit Dark Red (13.27 mg/g dry weight total pigment production). The ethanol extract, derived from roots of the same culture grown for 15 days under submerged conditions, showed a high antiradical activity (83% of inhibition of the stable DPPH·).

Betalain production in plant in vitro systems

Betalains have been widely used as natural colorants for many centuries, but their attractiveness for use as colorants of foods (or drugs and cosmetics) has increased recently due to their reportedly high anti-oxidative, free radical scavenging activities and concerns about the use of various synthetic alternatives. The main commercial sources of betalains are powders and concentrates of red beet (Beta vulgaris) or cactus pear (Opuntia ficus-indica) extracts. However, in recent years the technical and commercial feasibility of various in vitro systems to produce them biotechnologically has been explored. These research activities have included assessments of novel approaches for cultivating plant cell or tissue cultures, and diverse bioreactor systems for increasing production levels of secondary metabolites. This paper reviews recent progress in plant in vitro systems for producing betalain pigments. In addition, the factors that could be manipulated, the bioreactor systems that could be used, and the strategies that could be applied to improve betalain production are discussed.

Antioxidant activity of extracts from Lavandula vera MM cell cultures

Abstract The antioxidant activity of methanolic and ethyl acetate extracts from Lavandula vera MM cell culture were evaluated by the Schaal oven test in bulk sunflower oil and by the DPPH radical method. The oil oxidation was followed by measuring the quantity of primary oxidation products (peroxide value). Authentic rosmarinic acid, caffeic acid and BHT were tested in parallel for comparison. Ethyl acetate extract much better protected the oil from oxidation than methanolic extract and its antioxidant efficiency was comparable to that of pure rosmarinic and caffeic acids and much stronger than that of BHT. Both cell culture extracts and the authentic phenolic acids were much stronger scavengers of DPPH free radical than BHT on an equimolar basis.

Rosmarinic acid production by Lavandula vera MM cell-suspension culture

Abstract The time courses of growth and rosmarinic acid production by Lavandula vera MM cell suspension were investigated. The uptake of the main nutrients (sucrose, nitrogen, phosphorus, K, Ca, Mg) was followed during cultivation and the data on the physiology of the L. vera MM cell culture are presented. It was established that the cell culture synthesizes rosmarinic acid during the linear phase of growth for a relatively short period (between the 4th and 8th days of cultivation). The influence of sucrose concentration in the nutrient medium on cell growth and accumulation of rosmarinic acid by L. vera MM cell culture was investigated. The results showed that 7% sucrose in the nutrient medium ensured a steady growth of the cell suspension and increased the yield of rosmarinic acid (29.2 g/l dry biomass and 507.5 mg/l rosmarinic acid compared to 13.0 g/l dry biomass and 68.6 mg/l rosmarinic acid for the control cultivation with 3% sucrose).

Optimization of Rosmarinic Acid Production by Lavandula vera MM Plant Cell Suspension in a Laboratory Bioreactor

The all‐round effect of dissolved oxygen concentration, agitation speed, and temperature on the rosmarinic acid production by Lavandula veraMM cell suspension was studied in a 3‐L laboratory bioreactor by means of the modified Simplex method. Polynomial regression models were elaborated for description of the process of rosmarinic acid production (Y) in the bioreactor as a consequence of the variation of the dissolved oxygen (X1) concentration between 10% and 50%; agitation (X2) between 100 and 400 rpm; and temperature (X3) between 22 and 30 °C. The optimization made it possible to establish the optimal conditions for the biosynthesis of rosmarinic acid by L. veraMM: dissolved oxygen (X1*), 50% of air saturation; agitation (X2*), 400 rpm; and temperature (X3*), 29.9 °C, where maximal yield (Ymax) of 3489.4 mg/L of rosmarinic acid was achieved (2 times higher compared with the shake‐flasks cultivation).

Nutrient Medium Optimization for Rosmarinic Acid Production by Lavandula vera MM Cell Suspension

The overall effect of NH4NO3, KNO3, and KH2PO4 on the biosynthesis of rosmarinic acid and cell biomass by Lavandula vera MM cell suspension was studied by the method of the full factor experiment. Polynomial regression models were elaborated to give a quantitative description of the processes of biosynthesis of rosmarinic acid (Y1) and cell biomass (Y2) as a result of the variation of the concentration of NH4+, 0.09 g/L ≤ X1 ≤ 0.23 g/L; NO3‐, 2.44 g/L ≤ X2 ≤ 3.02 g/L; and KH2PO4, 0.170 ≤ X3 ≤ 0.425 g/L. Optimization procedures according to the modified Simplex method allowed us to establish the optimal conditions for the biosynthesis of rosmarinic acid by Lavandula vera MM: X1* = 0.09 g/L; X2* = 3.02 g/L, and X3* = 0.170 g/L, where Y1*max = 1786.74 mg/L (27 times higher compared with the cultivation in the standard Linsmayer‐Skoog medium). As a result, modified ingredients of the Linsmayer‐Skoog nutrient medium were applied for the cultivation of Lavandula vera MM to achieve a maximum yield of rosmarinic acid.

Galanthamine production by Leucojum aestivum L. shoot culture in a modified bubble column bioreactor with internal sections

Shoot culture of summer snowflake (Leucojum aestivum L.) was successfully cultivated in an advanced modified glass‐column bioreactor with internal sections for production of Amaryllidaceae alkaloids. The highest amounts of dry biomass (20.8 g/L) and galanthamine (1.7 mg/L) were achieved when shoots were cultured at 22°C and 18 L/(L·h) flow rate of inlet air. At these conditions, the L. aestivum shoot culture possessed mixotrophic‐type nutrition, synthesizing the highest amounts of chlorophyll (0.24 mg/g DW (dry weight) chlorophyll A and 0.13 mg/g DW chlorophyll B). The alkaloids extract of shoot biomass showed high acetylcholinesterase inhibitory activity (IC50 = 4.6 mg). The gas chromatography–mass spectrometry (GC/MS) profiling of biosynthesized alkaloids revealed that galanthamine and related compounds were presented in higher extracellular proportions while lycorine and hemanthamine‐type compounds had higher intracellular proportions. The developed modified bubble‐column bioreactor with internal sections provided conditions ensuring the growth and galanthamine production by L. aestivum shoot culture.

Purification of Rosmarinic Acid by Strong Ion‐Exchange Centrifugal Partition Chromatography

Abstract Ion‐Exchange centrifugal partition chromatography using benzalkonium chloride as a strong exchanger (SIXCPC) was successfully used to purify rosmarinic acid from a crude extract that was produced by callus culture. The purification process was carried out on a gram scale using the ternary biphasic system CHCl3: n‐BuOH∶water 4.5:1:4.5 v/v/v in the ascending mode (mobile aqueous phase and stationary organic phase). Two particular points are discussed: the influence of benzalkonium chloride on ternary solvent system stability and the advantage of injecting the analytes as sodium salts rather than molecular acids.

Rosmarinic acid from Lavandula vera MM cell culture

Abstract Rosmarinic acid was isolated as the main phenolic component of Lavandula vera MM cell culture. It was identified by means of TLC, HPLC, 1 H NMR, 13 C NMR and mass spectroscopy

Enhanced rosmarinic acid production by Lavandula vera MM cell suspension culture through elicitation with vanadyl sulfate

The influence of elicitation on rosmarinic acid biosynthesis by Lavandula vera MM cell suspension culture was investigated using vanadyl sulfate as an abiotic elicitor. It was established that 12 h after treatment with 25 mg/l vanadyl sulfate the rosmarinic acid production was increased up to 3.92 g/l (2.8 times higher compared to the control cultivation). No significant amounts of rosmarinic acid were detected in the culture medium in comparison with its intracellular content. However, it was observed that the extracellular content of rosmarinic acid is 3.3 times higher compared to the control variant (4 h after treatment at elicitor concentration 25 mg/l).