SUPPLEMENTARY MATERIAL Excelsanone, a new isoflavonoid from Erythrina excelsa (Fabaceae), with in vitro antioxidant and in vitro cytotoxic effects on prostate cancer cells lines. Joël AG. Yaya1,2*, Hadidjatou Daïrou1, Stephane Zingué3, Emmanuel Talla4*, Alembert T. Tchinda1, Michel Frédérich5, Joseph T. Mbafor2 1 Centre for Research on Medicinal Plants and Traditional Medicine, Institute of Medical Research and Medicinal Plants Studies, P.O. Box 13033, Yaounde, Cameroon 2 Department of Organic Chemistry, Faculty of Sciences, University of Yaounde 1, P.O. Box 812 Yaounde, Cameroon 3 Department of Life and Earth Sciences, Higher Teachers’ Training College, University of Maroua, P.O. Box 55 Maroua, Cameroon 4 Department of chemistry, Faculty of Sciences, University of Ngaoundere, P.O.Box 454, Ngaoundere, Cameroon 5 Laboratory of Pharmacognosy, Department of Pharmacy, CIRM, University of Liege, B36, 4000 Liege, Belgium Abstract A new isoflavonoid, excelsanone (2), was isolated from the ethyl acetate extract of Erythrina excelsa Backer stem bark, together with three known compounds namely 6,8diprenylgenistein (3), β-sitosterol (1) and sitosteryl-β-D-glucopyranoside (4). Their structures were elucidated using spectroscopic methods (HR-ESI-MS, NMR and IR) and by comparison with some data reported in literature. The antioxidant activity of crude extracts and two isolated compounds was evaluated using free radical scavenging (DPPH) and Ferric Reducing Ability Power (FRAP) methods with catechin as standard. The results of this radical scavenging activity showed that excelsanone (2) has a moderate potential with an IC50 of 1.31 mg/ml. The cytotoxicity of compounds 2 and 3 as well as the ethyl acetate extract was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in two prostate cancer cell lines (DU145 and PC3). Excelsanone (2) induced a greater cytotoxicity in all tested cell lines, with a significant inhibition of DU145 cells growth in a concentration-dependent manner. Keywords: Erythrina excelsa; Fabaceae; isoflavonoid; antioxidant, cytotoxicity 1 Contents: Table S1: 1H (500 MHz), 13C (125 MHz) NMR and HMBC data of excelsanone 1 in CDCl3 [δ (ppm), J (Hz)] Figure S1: Important HMBC ( (1) ) and 1H-1H COSY ( ) correlations of excelsanone Figure S2: ESIMS of excelsanone (2) Figure S3: IR spectrum of excelsanone (2) Figure S4: UV spectrum of excelsanone (2) Figure S5: 1H NMR spectrum (500 MHz, CDCl3) of excelsanone (2) Figure S6: APT spectrum (125 MHz, CDCl3) of excelsanone (2) Figure S7: HMBC spectrum (500 MHz: 1H, 125 MHz: 13C, CDCl3) of excelsanone (2) Figure S8: HSQC spectrum (500 MHz: 1H, 125 MHz: 13C, CDCl3) of excelsanone (2) Figure S9: 1H 1H COSY spectrum (500 MHz, CDCl3) of excelsanone (2) Table S2: DPPH and FRAP results Figure S10: DPPH graph of extracts Figure S11: DPPH graph of extracts and compounds 2 and 3 Figure S12: DPPH graph of extracts and control Figure S13: DPPH graph of extracts, compounds 2 and 3 and of the control Figure S14: DPPH graph of compounds 2 and 3 Figure S15: DPPH graph of compounds 2 and 3 and of the control Figure S16: Growth of DU145 and PC3 prostate carcinoma cells treated with different concentrations of compound 1 and 2 as well as E. excelsa ethyl acetate extract after 24, 48 and 72 hours. Controls remained untreated. (n = 3) 2 Table S1: 1H (500 MHz), 13C (125 MHz) NMR and HMBC data of excelsanone (1) in CDCl3 [δ (ppm), J (Hz)] N° δC (ppm) δH (ppm) HMBC 2 152.7 7.90 (1H, s) C (4, 3, 9) 3 123.7 4 181.6 5 157.9 6 127.8 5.63 (1H, d, 9.55 Hz) C(5, 9, 10) 7 115.9 6.74 (1H, d, 9.55 Hz) C (5, 8) 8 107.6 9 155.1 10 105.1 OH-5 13.08 (1H, s) 1’ 123.7 2’, 6’ 131.5 7.40 (1H, d, 8.86 Hz) C (4’, 3’, 3) 3’, 5’ 115.9 6.89 (1H, d, 8.86 Hz) C (4’, 3’, 2’) 4’ 155.8 1’’ 21.7 3.40 (2H, d, 7.85 Hz) C (8, 9, 2’’, 3’’, 4’’,5’’) 2’’ 122.1 5.18 (1H, t, 7.85) C (7, 9) 3’’ 132.2 4’’ 25.9 1.69 (3H, s) C (2”, 3’’,5’’) 5’’ 18.2 1.82 (3H, s) C (2’’,3’’,4’’) 3 Figure S1: Important HMBC ( (1) ) and 1H-1H COSY ( ) correlations of excelsanone Figure S2: ESIMS of excelsanone (1) 4 5 Figure S3: IR spectrum of excelsanone (2) Figure S4: UV spectrum of excelsanone (2) 6 Figure S5: 1H NMR spectrum (500 MHz, CDCl3) of excelsanone (2) Figure S6: APT spectrum (125 MHz, CDCl3) of excelsanone (2) 7 Figure S7: HMBC spectrum (500 MHz: 1H, 125 MHz: 13C, CDCl3) of excelsanone (2) 8 Figure S8: HSQC spectrum (500 MHz: 1H, 125 MHz: 13C, CDCl3) of excelsanone (2) Samples nBuEx EAEx AqRes Ex HEEx Excelsanone 2 DPPH IC 50 (mg/ml) 0.8096 ± 0.02 2.76 ± 0.06 1.069 ± 0.12 0.8303 ± 0.14 1,31 ± 0.04 6,8-diprenylgenistein 3 0.0760 ± 0.001 Catechin (μM/ml) Gallic Acid (ug/ml) 26.7 rum (500 MHz, CDCl3) of excelsanone (2) FRAP (mg/g of catechin eq) 170.82 ± 13.22 31.30 ± 2.31 33.61 ± 9.73 106.38 ± 21.28 150.29 ± 5.74 320.34 ± 3.07 Figu re S9: 1 H 1 H COS Y spect Table S2: Results of the antioxidant activity 9 100 R es aq Eex % In h ib itio n 80 HEex N bu 60 EAEx 40 20 0 0 1 2 3 4 5 C o n c e n t r a tio n m g /m l Figure S10: DPPH graph of extracts 100 R es aq Eex % In h ib itio n 80 HEex N bu 60 EAEx 40 6 ,8 -D ip r e n y lg e n is te in e x c e ls a n o n e 20 0 0 1 2 3 4 5 C o n c e n t r a tio n m g /m l Figure S11: DPPH graph of extracts and of compounds 2 and 3 10 100 R es aq Eex % In h ib itio n 80 HEex N bu 60 EAEx 40 g a llic a c id 20 0 0 1 2 3 4 5 C o n c e n t r a tio n m g /m l Figure S12: DPPH graph of extracts and control 100 R es aq Eex % In h ib itio n 80 HEex N bu 60 EAEx 40 6 ,8 -D ip r e n y lg e n is te in e x c e ls a n o n e 20 g a llic a c id 0 0 1 2 3 4 5 C o n c e n t r a tio n m g /m l Figure S13: DPPH graph of extracts, compounds 2 and 3 and of the control 100 6 ,8 - d ip re n y lg e n is te in % In h ib itio n 80 e x c e ls a n o n e 60 40 20 0 0 .0 0 .5 1 .0 1 .5 C o n c e n t r a tio n m g /m l Figure S14: DPPH graph of compounds 2 and 3 11 100 6 ,8 -D ip r e n y lg e n is te in % In h ib itio n 80 e x c e ls a n o n e g a llic a c id 60 40 20 0 0 .0 0 .5 1 .0 1 .5 C o n c e n t r a tio n m g /m l Figure S15: DPPH graph of compounds and of the control PC3 Cells DU145 Cells 240 260 Control EEx 10 µg/mL EEx 50 µg/mL EEx 100 µg/mL 220 Cell Number in % Cell Number in % 240 Control EEx 10 µg/mL EEx 50 µg/mL EEx 100 µg/mL 220 200 180 160 140 120 200 180 160 140 120 100 100 80 80 20 30 40 50 60 70 20 80 30 50 60 70 80 70 80 Incubation [h] Incubation [h] DU145 Cells PC3 Cells 240 260 Control (1) 1 µg/mL (1) 10 µg/mL (1) 20 µg/mL 200 Control (1) 1 µg/mL (1) 10 µg/mL (1) 20 µg/mL 240 Cell Number in % 220 Cell Number in % 40 180 160 140 120 100 80 220 200 180 160 140 120 100 60 80 20 30 40 50 60 Incubation [h] 70 80 20 30 40 50 60 Incubation [h] 12 DU145 Cells PC3 Cells 220 240 Control (2) 1 µg/mL (2) 10 µg/mL (2) 20 µg/mL 220 Cell Number in % Cell Number in % 200 Control (2) 1 µg/mL (2) 10 µg/mL (2) 20 µg/mL 180 160 140 120 100 200 180 160 140 120 100 80 80 20 30 40 50 60 70 80 Incubation [h] 20 30 40 50 60 70 80 Incubation [h] Figure S16: Growth of DU145 and PC3 prostate carcinoma cells treated with different concentrations of compound 2 and 3 as well as E. excelsa ethyl acetate extract after 24, 48 and 72 hours. Controls remained untreated. (n = 3). 13