NMR spectra (1H, 13C, 1H COSY, HMBC, and HMQC) were recorded on a JEOL JNM ECA 600 FT-NMR spectrometer (600 MHz for 1H and 150 MHz for 13C) using chloroform-d (99.8% D) as a solvent, with 0.03% tetramethylsilane (TMS) as an internal standard. Chemical shifts were expressed as δ (in ppm), and coupling constants (J) were recorded in Hertz.
The molecular weight of the compounds was analyzed by GC–MS after silylation of the samples, which was done with a QP2010 SE gas chromatograph mass spectrometer (Shimadzu, Tokyo, Japan). The device was equipped with an Intercap 5MS fused silica capillary column (30 mm × 0.25 mm I.D. and 0.25 µm film thickness), with an electronic pressure control module and a split/splitless injector (AOC 20i, Shimadzu). GC temperature was programmed from 100 to 310 °C at a rate of 5 °C/min, with helium as a carrier gas. The pressure was 100 kPa, split ratio was 1:80, the flow rate through the column was 1.33 mL/min in constant flow mode, and the ionization voltage was 70 eV. Approximately 1 mg of the propolis sample was mixed with 50 µL of dry pyridine and 75 µL of bis(trimethylsilyl) trifluoroacetamide. The mixture was heated at 80 °C for 20 min, and 1 µL of the sample was injected.
The molecular weight of the compounds was analyzed by GC–MS after silylation of the samples, which was done with a QP2010 SE gas chromatograph mass spectrometer (Shimadzu, Tokyo, Japan). The device was equipped with an Intercap 5MS fused silica capillary column (30 mm × 0.25 mm I.D. and 0.25 µm film thickness), with an electronic pressure control module and a split/splitless injector (AOC 20i, Shimadzu). GC temperature was programmed from 100 to 310 °C at a rate of 5 °C/min, with helium as a carrier gas. The pressure was 100 kPa, split ratio was 1:80, the flow rate through the column was 1.33 mL/min in constant flow mode, and the ionization voltage was 70 eV. Approximately 1 mg of the propolis sample was mixed with 50 µL of dry pyridine and 75 µL of bis(trimethylsilyl) trifluoroacetamide. The mixture was heated at 80 °C for 20 min, and 1 µL of the sample was injected.