To extract soluble PAs from tobacco seeds, 200 mg of dry seeds were ground in liquid nitrogen and extracted with 1 ml extraction solution (70% acetone/0.5% acetic acid) by vortexing for 10 s. After sonication at room temperature for 1 h, the mixture was centrifugation at 2,500 g for 10 min, and the residue was re-extracted twice. The pooled supernatants were extracted twice with hexane. To quantify the soluble PAs level, 50 µL of the supernatant sample was mixed with 200 µL of DMACA reagent (0.1% DMACA, 90% ethanol, 10% HCl) in 96-well plates and the absorption was measured at 640 nm. Soluble PA levels were calculated using a standard curve prepared using procyanidin B1 (Sigma, USA).
The residue from soluble PAs extraction was air dried and used for quantitative analysis of insoluble PAs. 500 µL butanol-HCl reagent (95% butanol: 5% concentrated HCl) was added to the residue and the mixture was sonicated at room temperature for 1 h, followed by centrifugation at 2,500 g for 10 min. The absorption of the supernatant was measured at 550 nm, then samples were boiled for 1 h, cooled to room temperature, and the absorbance at 550 nm was recorded again, with the rst value being subtracted from the second. Absorbance values were converted into PAs equivalents using a standard curve of procyanidin B1 (Sigma, USA).
The residue from soluble PAs extraction was air dried and used for quantitative analysis of insoluble PAs. 500 µL butanol-HCl reagent (95% butanol: 5% concentrated HCl) was added to the residue and the mixture was sonicated at room temperature for 1 h, followed by centrifugation at 2,500 g for 10 min. The absorption of the supernatant was measured at 550 nm, then samples were boiled for 1 h, cooled to room temperature, and the absorbance at 550 nm was recorded again, with the rst value being subtracted from the second. Absorbance values were converted into PAs equivalents using a standard curve of procyanidin B1 (Sigma, USA).