Alkaloid extraction

Nicotine alkaloids were extracted according to techniques developed by Häkkinen and his colleagues [39] with some modifications: To extract tobacco alkaloids, one gram fresh weight of BY-2 cells were dispersed in 2 ml of water. The mixture was basified with 3 ml 3.3% NH₄OH. To release cell content, cells were lysed by ultrasonication for 2 minutes by means of a high-efficiency ultrasound device (UP 100H, Hielscher, Teltow, Germany) pulsed with 0.5 s intervals using amplitude of 100%. The lysate was spun down for 15 min at 2100 ×g (Z 383 K, Hermle KG, https://www.hermle.de), and the supernatant was collected and extracted with 10 ml dichloromethane. The mixture was incubated for 30 min at ambient temperature on an orbital shaker (150 rpm). Subsequently, the polar dichloromethane layer was separated and collected through a 50-ml separation funnel. In order to improve the efficiency of extraction, this step was repeated. Precipitated proteins were separated by centrifugation from the collected polar phase at 2100 ×g for 15 min. In the next step, the clear lower phase was concentrated by a rotary evaporator (Büchi^® rotary evaporator Model R-205, http://www.buchi.com) under a reduced pressure of 550 mbar and a temperature of 40°C. After complete evaporation of dichloromethane, the extract was dissolved in 500 μl of 80% (v/v) methanol for HPLC analysis.