Extraction of anthraquinones and LC-MS analysis

Early- and late-stage of seed samples were extracted with methanol using sonication for 30 min at 60°C. After extraction, samples were centrifuged at 12,000 rpm for 3 min at 25°C and the supernatant was filtered with 0.2 μm Acrodisc^® MS Syringe Filters with WWPTFE membrane (Pall Corporation, Port Washington, NY, USA). Quantitative analysis of anthraquinones was performed by a Triple TOF 5600+ Spectrometer with a DuoSpray ion source (AB Sciex, Ontario, CA, USA) coupled with a Nexera X2 UHPLC (Shimadzu, Kyoto, Japan) equipped with binary solvent manager, sample manager, column heater, and photodiode array detector. UHPLC was performed on a ACQUITY UPLC®BEH C18 column (1.7 μm, 2.1 x 100 mm, Waters Corporation, Milford, USA) and mobile phases consisted of 5 mM ammonium acetate in water (eluent A) and 100% acetonitrile (eluent B). The gradient profile was as follows: 0–1 min, 20% B; 1–3.5 min, 10–30% B; 3.5–8 min, 30–50% B; 8–12 min, 50–100% B; 11–17 min, 100% B. The flow rate was 0.5 mL/min and five microliters of samples were injected. For detecting peaks from test samples, MS parameter in ESI-negative mode was used as follows: nebulizing gas, 50 psi; heating gas, 50 psi; curtain gas, 25 psi; desolvation temperature, 500°C; ion spray voltage floating, 4.5 kV.