Symmetry c18 column

Dried root samples after uniformly mixing two plants per pot were ground into powder using a mortar and pestle, and passed through a 40-mesh sieve. About 0.05 g of sample was weighed and extracted in 10 mL methanol/water (70:30) for 30 min in an ultrasonic bath at 25°C. The extract solution was cooled to a temperature of 25°C and filtered using a 0.45 μm filter. A 10–μL aliquot of the filtrate was subjected to separation via high–performance liquid chromatography, using a reverse phase C₁₈ symmetry column (4.6 mm × 250 mm, pore size 5 μm; Waters Corp., Milford, MA, United States). The mobile phase comprised a gradient of deionized water:phosphoric acid (100:0.05, v/v) and acetonitrile. The separation process was conducted in the gradient elution mode (Supplementary Table S1) at 25°C, at a flow rate of 1.0 mL/min. The eluted compounds were detected spectrophotometrically at 237 nm, using a 2998 PDA photodiode array detector. Glycyrrhizic acid and glycyrrhizin were purchased from China National Institutes for Food and Drug Control. Their stock solutions were diluted with 70% aqueous methanol to obtain solutions with an appropriate concentration for calibration purpose (Zhang et al., 2013 ).

The dried root samples of liquorice plants were ground into powder with a mortar and pestle and passed through a 40-mesh sieve. Approximately 0.05 g sample was weighed accurately and extracted in 10 mL methanol/water (70:30) for 30 min in an ultrasonic bath at 25°C. The extract solution was cooled to 25°C and filtered through a 0.45 μm filter. A 10-μL aliquot of the filtrate was subjected to separation by high–performance liquid chromatography using a reverse phase C₁₈ symmetry column (4.6 mm × 250 mm, pore size 5 μm; Waters Corp., Milford, MA, United States). The mobile phase comprised a gradient of deionized water:phosphoric acid (100:0.05, v/v) and acetonitrile. The separation was conducted in the gradient elution mode (Supplementary Table S1) at 25°C with a flow rate of 1.0 mL/min. The eluted compounds were detected spectrophotometrically at 237 nm using a 2998 PDA photodiode array detector. Standard substances of glycyrrhizic acid and glycyrrhizin were purchased from China National Institutes for Food and Drug Control. Their stock solutions were diluted with 70% aqueous methanol to appropriate concentrations for calibration purpose (Zhang et al., 2013 ).

Root materials were dried at 40°C for 2 days. An aliquot (0.1 g) of powdered root material (40 mesh) was extracted in 10 mL methanol/water (70:30) for 30 min in an ultrasonic bath at room temperature. The extract solution was cooled to room temperature and filtered through a 0.45 μm filter. A 10 μL aliquot of the filtrate was subjected to separation by high-performance liquid chromatography (HPLC) through a reverse phase C18 Symmetry^® column (4.0 mm × 250 mm, pore size of 3 μm; Waters Corp., Milford, MA, United States). The mobile phase comprised a gradient of deionized water: phosphoric acid (100: 0.020, V/V) and acetonitrile. The separation was operated in the gradient elution mode (Supplementary Table S1) at 25°C with a flow rate of 1.0 mL/min. Eluted compounds were detected spectrophotometrically at 276, 360, 248, and 370 nm using a 996 PDA photodiode array detector (Waters Corp., Milford, MA, United States). Glycyrrhizic acid, liquiritin, isoliquiritin, isoliquiritigenin, and licochalcone were purchased from China National Institutes for Food and Drug Control. Stock solutions were diluted with 70% aqueous methanol to appropriate concentrations for calibration purposes. (Zhang et al., 2013 )

Peptides were sequenced on a Synapt G2 HDMS (Waters, Milfords, MS, EUA) mass spectrometer coupled to a UPLC NanoAcquity system with 1D technology (Waters, Milfords, MS, EUA) and captured by a C18 Symmetry column (5 μm, 180 μm × 20 mm) (Waters, Milfords, MS, EUA). The peptides were separated by using a 2–90% acetonitrile gradient in 0.1% formic acid and an HSS T3 analytical column (1.8 μm, 75 μm × 100 mm) (Waters, Milfords, MS, EUA) with a flow of 300 μL min^− 1 for 120 min. The data were acquired on a Waters Synapt G2S Q-TOF mass spectrometer equipped with a NanoLockSpray (Waters, Milfords, MS, EUA). The experiments were performed in the HDMSE mode (data-independent analysis). The mass spectra were processed with the ProteinLynxGlobalServer (PLGS) software version 3.1. The proteins were identified by comparison to the Aspergillus UNIPROT database (207,966 proteins) [42 ]. The defined parameters were automatic tolerance for precursors and ion products, minimum of three corresponding ion fragments per peptide, minimum of seven corresponding ion fragments per protein, trypsin missed cleavage, carbamidomethylation as a fixed modification, oxidation of methionine as a variable modification, and 4% FDR peptide.