Epicatechin gallate

In order to quantify and identify concentration of allelochemicals in leaf litter samples, ≥98% pure catechin, epigallocatechin (EGC), epicatechin gallate (ECG), epicatechin (EC), epigallocatechin gallate (EGCG), taxifolin (TF), and protocatechuic acid (PCA) were purchased as external standards from Cayman Chemical (1180 E. Ellsworth Road Ann Arbor, MI, United States). Formic acid and LC-MS grade methanol were also purchased from Sigma-Aldrich (St. Louis, MO, United States). For a stock solution preparation, 1 mg of every standard was suspended in 1 ml of 99.9% methanol and 0.1% by volume of Formic acid. Serial dilutions were made using the same solvent as the stock solution, with a dilution range of 1.25–10 μg/ml in order to obtain a calibration curve. Each solution was distinctly injected with a 10-μl aliquot for HPLC-ESI-MS analysis. With all correlation coefficients > 0.998, excellent linearity was achieved within the calibration range (Supplementary Figures S1, S2).

The purity of all standards was >98%. Gallic acid, hydroxycinnamic acids (chlorogenic, caffeic, p-coumaric, and ferulic acids), the flavones luteolin, apigenin, luteolin-7-O-glucoside, and apigenin-7-O-glucoside, the flavonols quercetin (hydrate) and quercetin-3-O-rutinoside (rutin hydrate), the flavanones naringenin and diosmetin, the flavanols (±)-catechin (hydrate), (−)-epigallocatechin, (−)-epicatechin, and their gallate derivatives [(−)-catechin gallate, (−)-epicatechin gallate and (−)-epigallocatechin gallate], and the isoflavone daidzein were from Cayman Chemical Co. (Ann Arbor, MI, USA). The flavone diosmetin was from Extrasynthese Co. (Lyon, France). Methanol Chromasolv for HPLC was from Sigma Chemical (St. Louis, MO, USA).

Unless otherwise stated, the reagents used in this study were from Sigma Aldrich (Poole, Dorset, UK). Sodium citrate-containing vacutainers were from Greiner Bio-One. Paraformaldehyde solution was from Alfa Aesar. Epigallocatechin gallate (EGCG), epicatechin gallate (ECG), catechin gallate (CG), epicatechin (EC), catechin (C), gallocatechin gallate (GCG), epigallocatechin (EGC) and gallocatechin (GC) were from Cayman Chemicals (Michigan, USA).