3.4. HPLC Analysis of Vindoline and Catharanthine Content

After harvest, the leaves in the seedlings were freeze-dried and ground into powder (Jiuyang, modelJYL-C16V, Jinan, China). Then, 100 mg of the powder was extracted with 1 mL of 95% methanol for 60 min at room temperature and centrifuged at 10,000 rpm for 15 min. The supernatant was collected and used for the vindoline and catharanthine content determination.

The content of vindoline and catharanthine was determined by HPLC with a Poroshell 120 EC-C18 column (4.6 × 250 mm, 4 µm particle size) (Waldbronn, Germany). The mobile phase consisted of water (A) and methanol (B). The flow rate was 1 mL/min, the column temperature was 30 °C, and the injection volume was 10 μL. The gradient elution process began with the mobile phase A at 80% in the first 20 min, then decreased from 80% to 20% for the next 10 min, while in the following 10 min it increased from 20% to 80%, and maintained at 80% in the last 10 min. The detection wavelengths were 310 nm and 280 nm for vindoline and catharanthine, respectively. The concentrations of vindoline and catharanthine in the sample were calculated from standard curves of each, and they were expressed as milligram per gram of dry weight.