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Isolation of specialized metabolites<br … Isolation of specialized metabolites<br />
The leaf powder of rice (90 g) was extracted with 90 % methanol as described in a previous study (Matsuda et al. 2012). The extract was dissolved, suspended in water, and partitioned into a hexane and water layer. The water layer was subjected to ODS column chromatography and eluted with CH3OH–H2O (0:100 → 100:0 v/v; containing 0.05 % formic acid) to afford nine fractions (Fr.1–9). These fractions were purified using semipreparative HPLC performed under the following conditions: column, Cadenza CD-C18 or Unison UK-C18 columns, Imtakt 150 9 10 mm i.d.; particle size, 3 lm; solvents, water and methanol or acetonitrile, containing 0.1 %v/v formic acid; and flowrate, 3.0 mL/min. The following compounds were obtained: 1 (4.52 mg), 2 (12.69 mg), 3 (2.07 mg), 4 (2.57 mg), 5 (1.15 mg), 6 (0.94 mg), 7 (1.51 mg), 8 (1.23 mg), 9 (0.71 mg), 10(2.53 mg), 11 (1.63 mg), 12 (3.93 mg), 13 (2.74 mg), 14 (0.58 mg), 15 (0.96 mg), 16 (1.89 mg), 17 (0.22 mg), 18 (0.09 mg), 19 (0.65 mg), 20 (0.20 mg), 21 (0.28 mg), 22 (0.64 mg), 23 (1.04 mg), 24 (0.76 mg), 25 (2.31 mg), 26 (2.20 mg), 27 (2.04 mg), 28 (1.25 mg), 29 (0.64 mg), 30 (0.29 mg), 31 (0.63 mg), 32 (2.29 mg), 33 (4.84 mg), 34 (4.99 mg), 35 (1.13 mg), and 36 (2.70 mg). For details regarding the isolation procedures from rice, see Supplementary data file S1.<br />
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LC–quadrupole time-of-flight-tandem mass spectrometry (LC–QTOF-MS/MS) analysis<br />
LC analysis was performed on the Waters ACQUITY UPLCTM System. Samples were injected into an ACQUITY bridged ethyl hybrid (BEH) C18 column (100× 2.1 mm i.d.,1.7μm;Waters,Milford, MA, USA),and the column temperature was set at 40 ℃.The mobile phase consisted of A (0.1 % v/v formic acid in water) and B (0.1 % v/v formic acid in acetonitrile). The gradient conditions of the mobile phase were as follows: 0 min,99.5 % A; 10.0 min, 20 % A; 10.01 min, 0.5 % A;12.0 min, 0.5 % A; 12.1 min, 99.5 % A; and 14.5 min, 99.5 % A. The flow rate was 0.30 mL/min. UV–visible absorption spectra of samples were determined using a photodiode array (PDA) detector in the range of 200–600 nm. The sample injection volume was 1μL. <br />
MS detection was performed on a Waters Xevo G2 QTOF mass spectrometer with an electrospray ionization (ESI) interface (Waters). Full scan mass spectra were recorded through a range of 50–1,500 m/z. Nitrogen was used as the nebulizer and auxiliary gas; argon was utilized as the collision gas. The ESI source was operated in positive and negative ionization modes with a capillary voltage of 3 kV, sampling cone voltage of 25 V, cone gas flow of 50 L/h, desolvation gas flow of 800 L/h, desolvation temperature of 450 ℃, source temperature of 120 ℃, and CID energy ramped from 10 to 50 eV. Tandem MS analysis was performed using fast data directed analysis (FastDDA), which is rapid automated, intelligent MS/MS data acquisition for targeted qualitative analyses. isition for targeted qualitative analyses.
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