Supporting information C-Geranylated Flavanones from Paulownia tomentosa Fruits as Potential Anti-Inflammatory Compounds Acting via Inhibition of TNF-α Production Zuzana Hanáková, Jan Hošek, Petr Babula, Stefano Dall Acqua, Jiří Václavík, Karel Šmejkal, Department of Natural Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences Brno, Palackého tř. /, 6 Brno, Czech Republic Department of Molecular Biology and Pharmaceutical Biotechnology, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences Brno, Palackého tř. /, 6 Brno, Czech Republic Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Via F. Marzolo 5, 5 Padua, Italy Table of contents Figure S. H NMR ( MHz, MeD) spectrum of tomentodiplacone J () Figure S. HSQC( MHz, MeD) spectrum of tomentodiplacone J () Figure S. HMBC ( MHz, MeD) spectrum of tomentodiplacone J () Figure S. CSY ( MHz, MeD) spectrum of tomentodiplacone J () Figure S5. NESY ( MHz, MeD) spectrum of tomentodiplacone J () 5 Figure S6. H NMR ( MHz, MeD) spectrum of tomentodiplacone K () 5 Figure S7. HSQC ( MHz, MeD) spectrum of tomentodiplacone K () 6 Figure S8. HMBC ( MHz, MeD) spectrum of tomentodiplacone K () 6 Figure S. CSY ( MHz, MeD) spectrum of tomentodiplacone K () 7 Figure S. NESY ( MHz, MeD) spectrum of tomentodiplacone K () 7 Figure S. H NMR ( MHz, MeD) spectrum of tomentodiplacol B () 8 Figure S. HSQC ( MHz, MeD) spectrum of tomentodiplacol B () Figure S. HMBC ( MHz, MeD) spectrum of tomentodiplacol B () Figure S. CSY ( MHz, MeD) spectrum of tomentodiplacol B () Figure S5. NESY( MHz, MeD) spectrum of tomentodiplacol B () Figure S6. H NMR ( MHz, MeD) spectrum of, --dimethyl-5 -hydroxydiplacone () Figure S7. HSQC ( MHz, MeD) spectrum of, --dimethyl-5 -hydroxydiplacone () Figure S8. HMBC ( MHz, MeD) spectrum of
, --dimethyl-5 -hydroxydiplacone () Figure S. CSY ( MHz, MeD) spectrum of, --dimethyl-5 -hydroxydiplacone () Figure S. NESY ( MHz, MeD) spectrum of, --dimethyl-5 -hydroxydiplacone () Figure S. H NMR ( MHz, MeD) spectrum of mimulone F (5) Figure S. HSQC ( MHz, MeD) spectrum of mimulone F (5) Figure S. HMBC ( MHz, MeD) spectrum of mimulone F (5) Figure S. CSY ( MHz, MeD) spectrum of mimulone F (5) 5 Figure S5. NESY ( MHz, MeD) spectrum of mimulone F (5) 5 Figure S6. H NMR ( MHz, MeD) spectrum of mimulone G (6) 6 Figure S7. HSQC ( MHz, MeD) spectrum of mimulone G (6) 6 Figure S8. HMBC ( MHz, MeD) spectrum of mimulone G (6) 7 Figure S. CSY ( MHz, MeD) spectrum of mimulone G (6) 7 Figure S. NESY ( MHz, MeD) spectrum of mimulone G (6) 8 Figure S. H NMR ( MHz, MeD) spectrum of mimulone H (7) 8 Figure S. HSQC ( MHz, MeD) spectrum of mimulone H (7) Figure S. HMBC ( MHz, MeD) spectrum of mimulone H (7) Figure S. CSY ( MHz, MeD) spectrum of mimulone H (7) Figure S5. NESY ( MHz, MeD) spectrum of mimulone H (7) Figure S6. H NMR ( MHz, MeD) spectrum of paulownione A (8) Figure S7. HSQC ( MHz, MeD) spectrum of paulownione A (8) Figure S8. HMBC ( MHz, MeD) spectrum of paulownione A (8) Figure S. CSY ( MHz, MeD) spectrum of paulownione A (8) Figure S. H NMR ( MHz, MeD) spectrum of paulownione B () Figure S. HSQC ( MHz, MeD) spectrum of paulownione B () Figure S. HMBC ( MHz, MeD) spectrum of paulownione B () Figure S. CSY ( MHz, MeD) spectrum of paulownione B () Figure S. NESY ( MHz, MeD) spectrum of paulownione B () 5 Figure S5. H NMR ( MHz, MeD) spectrum of tomentodiplacone L () 5 Figure S6. HSQC ( MHz, MeD) spectrum of tomentodiplacone L () 6 Figure S7. HMBC ( MHz, MeD) spectrum of tomentodiplacone L () 6 Figure S8. CSY ( MHz, MeD) spectrum of tomentodiplacone L () 7 Figure S. NESY ( MHz, MeD) spectrum of tomentodiplacone L () 7 Figure S5. H NMR ( MHz, MeD) spectrum of tomentone () 8 Figure S5. HSQC ( MHz, MeD) spectrum of tomentone () 8 Figure S5. HMBC ( MHz, MeD) spectrum of tomentone () Figure S5. CSY ( MHz, MeD) spectrum of tomentone () Figure S5. NESY ( MHz, MeD) spectrum of tomentone () Figure S55. H NMR ( MHz, CDCl ) spectrum of,,5 -trimethoxyflavanone () Figure S56. HSQC ( MHz, CDCl ) spectrum of,,5 -trimethoxyflavanone () Figure S57. HMBC ( MHz, CDCl ) spectrum of,,5 -trimethoxyflavanone () Figure S58. CSY ( MHz, CDCl ) spectrum of,,5 -trimethoxyflavanone () Figure S5. NESY ( MHz, CDCl ) spectrum of,,5 -trimethoxyflavanone () Figure S6. The influence of selected flavanones on the degradation of IκB-α Table S. IC 5 values of compounds 5
....5....8...8.7..6.5.8..6.5.5.555.65.7..6.58.75.7.7.77.75...55.68.7.58..6...8.6.5.88.865.88.8.78.88 5.5 5.6 5.78 5.8 6. 6.8 6.8 6.6 6. 7.8 H NMR (CD D, MHz) H H H 8 7 6 5 Figure S. H NMR ( MHz, MeD) spectrum of tomentodiplacone J () H H H 6 8 6 8 7 6 5 Figure S. HSQC( MHz, MeD) spectrum of tomentodiplacone J ()
H H H 6 8 6 8 8 7 6 5 Figure S. HMBC ( MHz, MeD) spectrum of tomentodiplacone J H H H 5 6 7 8 8 7 6 5 Figure S. CSY ( MHz, MeD) spectrum of tomentodiplacone J ()
.8.8.....6......8.5.8.8..7.6.67.6.7.7.7.86.76.7.75.757.77.7..5.7.58.5...68.8.866.88.8..8.776.887.5 5.78 5. 5. 5.55 5.6 5.7 6.8 6.8 6. 6.6 6.5 7.87 7. H H H Figure S5. NESY ( MHz, MeD) spectrum of tomentodiplacone J () H NMR (CD D, MHz) H H H H 8 7 6 5 Figure S6. H NMR ( MHz, MeD) spectrum of tomentodiplacone K () 5
H H H H 6 8 6 8 7 6 5 Figure S7. HSQC ( MHz, MeD) spectrum of tomentodiplacone K () H H H H 6 8 6 8 8 7 6 5 Figure S8. HMBC ( MHz, MeD) spectrum of tomentodiplacone K () 6
H H H H 5 6 7 8 8 7 6 5 Figure S. CSY ( MHz, MeD) spectrum of tomentodiplacone K () H H H H Figure S. NESY ( MHz, MeD) spectrum of tomentodiplacone K () 7
..5.5..88..........8..8.8.5....58.66.685.7....76..5...7.877.88.87.88.8.65.6.6.68.888 5. 5.5 5.567 5. 6.657 6.68 6.85 6.865 6.8 6.8 7.5 7. 7.6 8.57 H NMR (CD D, MHz) H H H 8 7 6 5 Figure S. H NMR ( MHz, MeD) spectrum of tomentodiplacol B () 8
H H H 6 8 6 8 7 6 5 Figure S. HSQC ( MHz, MeD) spectrum of tomentodiplacol B () H H H 6 8 6 8 8 7 6 5 Figure S. HMBC ( MHz, MeD) spectrum of tomentodiplacol B ()
H H H 5 6 7 8 8 7 6 5 Figure S. CSY ( MHz, MeD) spectrum of tomentodiplacol B () H H H Figure S5. NESY( MHz, MeD) spectrum of tomentodiplacol B ()
....6...8.8...5.75.76.7....56.57.67.66.67.7.767.768.787.5.6.8..5.78.7.76.7.75.766.5.57.68.....5...7.68.8.867.886 5.6 5.75 5.7 5.8 5.6 5. 5.7 5. 5.6 5. 5.87 5. 5.8 5.5 5.86 6.8 6.66 6.6 6.655 H NMR (CD D, MHz) H H H 8 7 6 5 Figure S6. H NMR ( MHz, MeD) spectrum of, --dimethyl-5 -hydroxydiplacone () H H H 6 8 6 8 7 6 5 Figure S7. HSQC ( MHz, MeD) spectrum of, --dimethyl-5 -H-diplacone ()
H H H 6 8 6 8 8 7 6 5 Figure S8. HMBC ( MHz, MeD) spectrum of, --dimethyl-5 -H-diplacone () H H H 5 6 7 8 8 7 6 5 Figure S. CSY ( MHz, MeD) spectrum of, --dimethyl-5 -H-diplacone ()
...8..7.........7.58.6..6.7..5.6.7.75.7.76.7.7.76.7...5.67.678.7.7.7.5.5.7....56.6..5...7.8 5.68 5.8 5.86 5. 5.7 5. 5. 5.6 6.8 6.85 6.87 6.8 6.8 7.8 7.5 7. 7. 7.6 7. H H H Figure S. NESY ( MHz, MeD) spectrum of, --dimethyl-5 -H-diplacone () H NMR (CD D, MHz) H H H H 5 H 8.5 8. 7.5 7. 6.5 6. 5.5 5..5..5..5..5..5. Figure S. H NMR ( MHz, MeD) spectrum of mimulone F (5)
H H H H 5 H 6 8 6 8 7 6 5 Figure S. HSQC ( MHz, MeD) spectrum of mimulone F (5) 6 8 6 8 8 7 6 5 Figure S. HMBC ( MHz, MeD) spectrum of mimulone F (5)
H H H H 5 H 5 6 7 8 8 7 6 5 Figure S. CSY ( MHz, MeD) spectrum of mimulone F (5) H H H H 5 H Figure S5. NESY ( MHz, MeD) spectrum of mimulone F (5) 5
..8.8.5.7..7...5.8.6.5.8...85.6....5.8.5.66.68.785.8.....66.67.76.7.55.5.88..8....7.77..8.58..5...7.68.88..7 5.66 5.8 5. 5.6 5. 5.8 6.88 6.8 6.85 6.8 7. 7.7 7. 7. 7. H NMR (CD D, MHz) H H H 6 H 8.5 8. 7.5 7. 6.5 6. 5.5 5..5..5..5..5..5. Figure S6. H NMR ( MHz, MeD) spectrum of mimulone G (6) H H H 6 H 6 8 6 8 7 6 5 Figure S7. HSQC ( MHz, MeD) spectrum of mimulone G (6) 6
6 8 6 8 8 7 6 5 Figure S8. HMBC ( MHz, MeD) spectrum of mimulone G (6) H H H 6 H 5 6 7 8 8 7 6 5 Figure S. CSY ( MHz, MeD) spectrum of mimulone G (6) 7
...6.5..6.....7..6..87...6.78...67.67.7.75.77.8.87.688.76.755.75.77.777.7.7.8.8..6..56.6.7.86.8.8.6..5...7.68.8.876.6.75.88.776.87.56 5.7 5. 5. 5.5 5.5 6.8 6.8 7. 7. 8.566 H H H 6 H Figure S. NESY ( MHz, MeD) spectrum of mimulone G (6) H NMR (CD D, MHz) H H H 7 H 8.5 8. 7.5 7. 6.5 6. 5.5 5..5..5..5..5..5. Figure S. H NMR ( MHz, MeD) spectrum of mimulone H (7) 8
H H H 7 H 6 8 6 8 7 6 5 Figure S. HSQC ( MHz, MeD) spectrum of mimulone H (7) 6 8 6 8 8 7 6 5 Figure S. HMBC ( MHz, MeD) spectrum of mimulone H (7)
H H H 7 H 5 6 7 8 8 7 6 5 Figure S. CSY ( MHz, MeD) spectrum of mimulone H (7) H H H 7 H Figure S5. NESY ( MHz, MeD) spectrum of mimulone H (7)
...5. 6.8..6...8...5.6.5...6.77..8.6.8...5.6.75..6.6.75.75.7.77.7.8..8..8.8.56...7.677.7.7.8...56..6..5...7.68.8.85.88.887 5.75 5. 5.5 5. 5. 6.75 6.8 H NMR (CD D, MHz) H H H H 8 H 8.5 8. 7.5 7. 6.5 6. 5.5 5..5..5..5..5..5. Figure S6. H NMR ( MHz, MeD) spectrum of paulownione A (8) H H H H 8 H 6 8 6 8 7 6 5 Figure S7. HSQC ( MHz, MeD) spectrum of paulownione A (8)
6 8 6 8 8 7 6 5 Figure S8. HMBC ( MHz, MeD) spectrum of paulownione A (8) H H H H 8 H 5 6 7 8 8 7 6 5 Figure S. CSY ( MHz, MeD) spectrum of paulownione A (8)
..8.8. 6.7.6....5.6.7...7.8....8.75........5.6.65.67.687.78....8.55.76.....678.7..5.56.6.76.8..57..5...7..5.68.7..5.885.88. 5.75 5. 5. 5.7 5. 6.75 8.57 H NMR (CD D, MHz) H H H H 8.5 8. 7.5 7. 6.5 6. 5.5 5..5..5..5..5..5. Figure S. H NMR ( MHz, MeD) spectrum of paulownione B () H H H H 6 8 8 7 6 5 Figure S. HSQC ( MHz, MeD) spectrum of paulownione B ()
H H H H 6 8 6 8 8 7 6 5 Figure S. HMBC ( MHz, MeD) spectrum of paulownione B () H H H H 5 6 7 8 8 7 6 5 Figure S. CSY ( MHz, MeD) spectrum of paulownione B ()
.......5.5..8....6.....66.77..5.575.6.65.65.66.68.6.75.8.5.6.77.56.5.57.558.576.7.75.87.8.8.868.88.8..8..5...7.68.86.87.85.87.885.8. 5.5 5. 5.8 5.8 5.6 5. 5.6 6.85 6.85 6. 6. 6.57 7.78 H H H H Figure S. NESY ( MHz, MeD) spectrum of paulownione B () H NMR (CD D, MHz) H H H 8.5 8. 7.5 7. 6.5 6. 5.5 5..5..5..5..5..5. Figure S5. H NMR ( MHz, MeD) spectrum of tomentodiplacone L () 5
H H H 6 8 6 8 7 6 5 Figure S6. HSQC ( MHz, MeD) spectrum of tomentodiplacone L () 6 8 6 8 8 7 6 5 Figure S7. HMBC ( MHz, MeD) spectrum of tomentodiplacone L () 6
H H H 5 6 7 8 8 7 6 5 Figure S8. CSY ( MHz, MeD) spectrum of tomentodiplacone L () H H H Figure S. NESY ( MHz, MeD) spectrum of tomentodiplacone L () 7
....7..7......7..8.7..8.8.6...66.8...58.6.667.7.76.77.76.87.875.85.7....75.6.7.765.87.88.5.86..56.6...6.8.88.68.6.85 5.7 5. 5.7 5.8 5.5 5.5 5.65 5. 5.7 6.6 6.65 6.87 6.5 8.565 H NMR (CD D, MHz) H H H 8.5 8. 7.5 7. 6.5 6. 5.5 5..5..5..5..5..5. Figure S5. H NMR ( MHz, MeD) spectrum of tomentone () H H H 6 8 6 8 7 6 5 Figure S5. HSQC ( MHz, MeD) spectrum of tomentone () 8
6 8 6 8 7 6 5 Figure S5. HMBC ( MHz, MeD) spectrum of tomentone () H H H 5 6 7 8 8 7 6 5 Figure S5. CSY ( MHz, MeD) spectrum of tomentone ()
....7. 6..7.8.6.86.85.877.887.5..6.6.7.5..5..7..5.6.6.....6.8.7.66.5...5.7.68.8.56.6.7.86.8.8.8..57.6.7 5.5 5.66 5.6 5.7 5. 6. 6.67 6.685 7. 7.8 7.5.5 Figure S5. NESY ( MHz, MeD) spectrum of tomentone () H NMR (CDCl, MHz) CDCl H H 8 7 6 5 Figure S55. H NMR ( MHz, CDCl ) spectrum of,,5 -trimethoxyflavanone ()
H H 6 8 6 8 7 6 5 Figure S56. HSQC ( MHz, CDCl ) spectrum of,,5 -trimethoxyflavanone () 6 8 6 8 8 7 6 5 Figure S57. HMBC ( MHz, CDCl ) spectrum of,,5 -trimethoxyflavanone ()
5 6 7 8 8 7 6 5 Figure S58. CSY ( MHz, CDCl ) spectrum of,,5 -trimethoxyflavanone () Figure S5. NESY ( MHz, CDCl ) spectrum of,,5 -trimethoxyflavanone ()
Figure S6. The influence of selected flavanones on the degradation of IκB-α. THP- macrophages were pre-treated with compounds, 7,,,, and prednisone (Pred.) at a concentration of µm or with DMS only (Veh. and CTRL) for h. Subsequently, LPS ( µg/ml) was added to trigger the IκB-α degradation. After min, the cells were collected and the amounts of IκB-α and β-actin were detected by Western blot analysis. The intensity of the digested bands was analysed by densitometric analysis. The blots show results representative of three independent experiments. The results are expressed as means ± S.E. for three independent experiments.
Table S. IC 5 values for compounds 5 IC5 6.7 ±. μm 6.5 ±. μm 6% * 6. ±. μm 5 6. ±.8 µm 6 8. ±.7 µm 7. ±. μm 8 8% * 87% * 6.7 ±. μm 6. ±.6 µm 78% * 8. ±. μm 7. ±. μm 5 8.6 ±. µm 6 8% * 7 > μm a 8 8.6 ±. µm 6.6 ±.6 μm 6. ±. µm 7. ±. μm > μm b 7. ±.5 μm 7% * 5 78% * * viability at µm a - DI:./j.76-58..7.x b - DI:./np76w