Mestrenova v 8

The chemical structure of β-carotene isolated from S. obliquus was confirmed by using ¹H-NMR analysis on an NMR spectrometer (500 MHz, JEOL, USA) using CDCl₃ (Merck, Germany) as the solvent. The chemical shifts are reported in ppm (parts per million; δ) and coupling constants (J) are expressed in Hz. TMS was used as an internal standard. For improving the signal-to-noise ratio. The data were analyzed using the software program MestReNova v8.0.2 (2012 Mestrelab Research S. L.).

The ¹H- and ¹³C-NMR (500 MHz, 125 MHz) spectra of P1 (isolated from S. obliquus) were recorded on an NMR spectrometer (JEOL, USA) with CDCl₃ as the solvent. The chemical shifts are reported in ppm (parts per million; δ) and coupling constants (J) are expressed in Hz. TMS was used as an internal standard. For improving the signal-to-noise ratio, total scans of 128 and 725 were performed for ¹H- and ¹³C-NMR, respectively. The data were analyzed using the software program MestReNova v8.0.2 (2012 Mestrelab Research S. L.).

All NMR spectra were recorded on a DRX 500-MHz NMR spectrometer (Bruker, Billerica, MA, USA) using a 5-mm BBO probe with a z-gradient. Hydroxyprotoilludene samples were dissolved in deuterated dichloromethane to a final concentration of ~ 50 mM and were calibrated against the solvent signals at 5.32 and 54.00 ppm. ¹³C-NMR spectra was acquired by conducting 1000 scans with a 30° pulse and a delay of 2 s. ¹³C-DEPT-135 spectra was also acquired by conducting 400 scans. All 2D spectra were acquired using Bruker standard pulse programs. The DQF-COSY and gs-NOESY spectra were measured in 512 increments, with two or four scans per increment, respectively. A mixing time of 600 ms was used for the acquisition of the NOESY spectrum. For heteronuclear correlation, the gs-HSQC and gs-HMBC spectra were measured in 512 increments, with four or eight scans per increment, respectively. The spectra were optimized for ¹J and ⁿJ couplings of 140 and 8 Hz, respectively. The data were processed using TopSpin v1.3 (Bruker) and MestReNova v8.0 (MestreLab Research, Santiago de Compostela, Spain).