ECHINACEA PURPUREA: HAIRY ROOT CULTURES FOR CAFFEIC ACID DERIVATIVES PRODUCTION

Echinacea products have gained considerable attention in the commercial area as general immune system enhancer. Among Echinacea species, the natural products with medicinal bioactivity are several caffeic acid derivatives (CADs), which are also strong antioxidants, and one of which, cichoric acid, possesses potential against HIV-1 integrase. Agrobacterium rhizogenes-mediated transformed hairy roots hold the potential for economically feasible biotechnological routes to the controlled biosynthesis of complex, plant-derived and natural molecules. A great deal of research work focused on the phytochemical characterization and pharmacological activity of Echinacea has been conducted; however, limited information is available regarding hairy root culture for consistent production of CADs. Inoculation of leaf explants of Echinacea purpurea (Moench) with Agrobacterium rhizogenes induced hairy roots with the capacity to produce biologically active caffeic acid derivatives (CADs), especially cichoric acid. The kinetics of growth, the uptake of macronutrients, and the accumulation of CADs were investigated in the cultured hairy roots for a 50-day period. Maximum biomass of 12.2 g/l was obtained at day 40, and mathematical relationship between hairy root growth and ionic conductivity was established during exponential phase. HPLC analyses of methanolic (0.1 % phosphoric acid; 70:30, v/v) extracts from hairy roots revealed the accumulation of CADs: cichoric acid (19.21 mg/g DW), caftaric acid (3.56 mg/g DW) and chlorogenic acid (0.93 mg/g DW).Continuous light exposure affected the magnitude of CADs biosynthesis in the hairy root cultures, and increased cichoric acid, caftaric acid and chlorogenic acid to 27.0 mg/g DW, 6.3 mg/g DW and 2.3 mg/g DW, respectively. Light-grown hairy roots also accumulated anthocyanins, which became visible in outer cell layer of the cortex as a ring of purple color. The growth rate and cell viability of the hairy root cultures in light did not show obvious difference in comparison with that in dark. The enhanced accumulation of CADs and anthocyanins was correlated to light-stimulated phenylalanine ammonium lyase (PAL) activity. Enhanced levels of malondialdehyde (MDA) and hydrogen peroxide (H2O2) in lightgrown purple hairy roots indicated that light caused oxidative stress. Antioxidants in root extracts from light- and dark-grown cultures were identified, and activities quantified using enzymatic and α, α-diphenyl-β-picrylhydrazyl (DPPHº) assays. Purple morphs in hairy roots held greater amounts of superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APx, EC 1.11.1.11), lignin (LN), total CADs and peroxidase (POD, EC 1.11.1.7). They were considerably more effective at scavenging DPPHº radicals, and also showed enhanced differentiation. Light- and dark-grown cultures did not show significant differences for glutathione peroxidase (GPx, EC 1.15.1.1) levels. Significantly higher levels of nitric oxide (NO) also contributed to antioxidant pool in light-grown cultures. It shows that hairy root cultures could grow under continuous light stress and protect themselves from toxicity of reactive oxygen species (ROS) by altering CADs levels and antioxidant enzymes. Exogenously applied gibberellic acid (GA3) has shown diverse effects on the growth, secondary metabolism and antioxidative defense system of light-grown hairy roots. Significant increase in biomass was obtained with moderate concentrations of GA3 ranging from 0.01 to 0.1 μM, and the treatment with 0.025 μM produced the highest amount of cichoric acid (36.2 mg/g DW), caftaric acid (7.8 mg/g DW) and chlorogenic acid (7.9 mg/g DW). Four concentrations of GA3 (control, 0.005, 0.025 and 1.0 μM) were selected tofurther study their impact on PAL, cell viability, hairy root morphology and anatomy and antioxidant enzymes. All of the activities determined were enhanced in 0.025 μM GA3 treatment whereas the opposite was observed with 0.005 and 1.0 μM levels of GA3. GA3 also affected root morphogenesis and radially thick, dense purple roots were observed at moderate concentrations and thin off-white roots were observed at higher and lower concentrations. Fluctuations in antioxidative system leads to conclusion that growth and CADs production is interrelated to antioxidative enzymes and is responsible for diverse effects observed in morphology and anatomy. Moreover, it is inferred that higher concentration of GA3 alleviated light-induced stress and alter growth phase. The hairy roots were grown in 2-l airlift bioreactor (1.7-l of working volume) with mesh for scaled-up CADs production. Mesh size affected hairy root distribution, growth and CADs accumulation with mesh 40 (700 μm pore size) being optimum for biomass and CADs production. 0.012 m3/h of air flow rate was found to be the best to accumulate dry biomass (15.2 g/bioreactor) and CADs (cichoric acid 16.4 mg/g DW; caftaric acid 3.1 mg/g DW and chlorogenic acid 0.59 mg/g DW). Ultrasonication (US) of root cultures in the airlift bioreactor resulted in considerably higher biomass accumulation and 6 minute exposure produced dry biomass of 21.8 g/bioreactor. US exposure inhibited CADs accumulation, but enhanced PAL activity. Further investigations revealed enhancement of anthocyanins and lignin by US, which were also PAL-derived metabolites. This thesis reports establishment of hairy root cultures with the ability to produce CADs, especially cichoric acid. Systematic approach was adopted to enhance CADs by light and GA3 in Erlenmeyer flask system. Furthermore, this study provides an effective approach for feasible production of CADs in airlift bioreactor. This hairy root system is exemplary due to its genetic and biochemical stability, and provides evidence that biotechnological production of CADs in hairy roots is possible