SUPPLEMENTARY MATERIAL Chemical composition, antioxidant and antibacterial properties of Pteranthus dichotomus from Algerian Sahara Zina Allaouaa, Mohammed Benkhaleda, Ammar Dibia, Christophe Longb, Mohammed Cherif Aberkanea, Soumia Bouzidic, Ahmed Kassah-Laouard and Hamada Habaa* a Laboratoire de Chimie et Chimie de l’Environnement (L.C.C.E), Département de Chimie, Faculté des Sciences, Université de Batna, Algérie; bUSR 3388 CNRS-Pierre Fabre, 3 Avenue Hubert Curien BP 13562, 31035 Toulouse, France; cLaboratoire de Biotechnologie des molécules bioactives et de la physiopathologie cellulaire, Département de biologie, Université de Batna, Algérie; dLaboratoire central de microbiologie CHU, Faculté de Médecine Université de Batna, Algérie * Corresponding author: Haba Hamada Tel: +213 33 86 89 86; fax: +213 33 86 89 86; E-mail address: haba.hamada@yahoo.fr The phytochemical study of ethyl acetate and n-butanol extracts of Pteranthus dichotomus Forssk. led to the isolation and identification of eleven compounds, including three glycolipids 1–3, one lignan 4 , three flavonoids 5–7 and four phytosterols 8–11. Structures of the isolated compounds have been elucidated by analysis of 1D and 2D NMR data, and mass spectrometry EI-MS and ESI-MS and by comparison with literature data. Furthermore, the ethyl acetate and n-butanol extracts were examined for their antioxidant and antibacterial activities. The results showed that both extracts (PDAC and PDBU) had a moderate antioxidant activity (IC50 = 375.514 μg/mL and 691.333 μg/mL) respectively. Keywords: Caryophyllaceae, Pteranthus dichotomus, glycolipids, flavonoids, NMR, ESI, antioxidant activity, antibacterial activity. 1 Experimental details 1. General experimental procedures UV spectra were measured on a Kontron UVS900 lite, Uvikon 941 spectrophotometer (Bio Tek instruments, USA). IR spectra were obtained using a Shimadzu model IR-470 spectrometer (Shimadzu, Ontario, Canada). 1H and13C NMR spectra were recorded in CD3OD and CDCl3 using a Bruker Avance II spectrometer (Bruker, France) equipped with a cryoprobe at 500 MHz for 1H and 125 MHz for 13 C. Complete assignments were performed on the basis of 2D experiments (COSY, HSQC and HMBC). Positive and negative ESI mass spectra were produced on ion trap Bruker Esquire. EI-MS were recorded using Maldimicro MX, micromasse walter. ESI-MS spectra were performed on a Bruker Micromass Q-TOF. Optical rotations were measured on a Perkin-Elmer 241 polarimeter (Perkin-Elmer, USA). Analytical TLC was performed in silica gel plates Kieselgel 60 F254 (Merck, Darmstadt, Germany) and developed by spraying with 50 % sulfuric acid reagent followed by heating. Column chromatography (CC) was performed using Merck Kieselgel 60 (70–230 mesh), Polyamide SC6, Sephadex LH-20 and lobar LiChroprep RP-8 (40–68 μm; Merck). The antioxidant activity was evaluated using spectrophotometer (Vis-7220G & UV-9200-UK). 2. Plant material Pteranthus dichotomus Forssk. was collected in May 2010 from the region of Biskra (Algerian Sahara) and was identified by Prof. Bachir Oudjehih of the agronomic department of Batna University where a voucher specimen has been deposited (No.710 / LCCE). 3. Extraction, fractionation and isolation The powdered aerial parts of Pteranthus dichotomus (600 g) were macerated in MeOH-H2O (80:20) (6 Liters × 2) for 3 days at room temperature. Filtration and evaporation of the extract gave an aqueouse-alcoholic solution. The aqueous phase was extracted successively with petroleum ether, ethyl acetate and n-butanol (each 300 mL x 3). The organic phases were dried with Na2SO4. After drying, filtration and evaporation the following extracts were obtained: petroleum ether PDPE (4 g), ethyl acetate PDAC (6.5 g), and n-butanol PDBU (15 g). 6 g of ethyl acetate extract (PDAC) were separated on vacuum liquid chromatography VLC (50 mm × 50 mm; fractions of 100 mL) using silica gel as stationary phase. The elution was carried out with a mixture of pertoleum ether–EtOAc and then EtOAc–MeOH to give 10 fractions Fr1 to Fr10. Fractions Fr1+2 (57 mg) were chromatographed on CC of silica gel eluting with petroleum ether–EtOAc (100:0 to 60:40) to yield 4 mg of compound 10 and 7 mg of a 2 mixture of two compounds 8 and 9. Fraction Fr4 (966 mg) was purified by silica gel column chromatography CC using CHCl3–MeOH (100:0 to 70:30) and afforded 10 subfractions (Fr4.1 to Fr4.10). Fr4.3 (44 mg) was purified by gel filtration on Sephadex LH-20 with CH2Cl2 as eluent to give 21.3 mg of compound 4. Fraction Fr8 (1200 mg) was filtered on Sephadex and provided 4 subfractions (Fr8.1 to Fr8.4). Fr8.2 (800 mg) was further chromatographed on a silica gel CC eluting with CHCl3–MeOH (100:0 to 70:30) to yield 10 subfractions (Fr8.2.1 to Fr8.2.10). From fraction Fr8.2.5 (400 mg), 4.5 mg of pure compound 11 were obtained by precipitation in MeOH. Fr8.2.10 (70 mg) was subjected to CC of polyamide eluting with toluene–MeOH (100:0 to 80:20) and afforded two compounds 6 (4 mg) and 5 (3.2 mg). The n-butanol extract PDBU (7.5 g) was submitted to vacuum liquid chromatography VLC (50 mm × 50 mm; fractions of 100 mL) on RP-8 using H2O–MeOH (80:20 to 0:100) to obtain 14 fractions of 100 mL combined together into 6 total fractions. After repeated polyamide CCs eluted with a gradient of toluene–methanol on fraction Fr2 (882 mg) followed by filtration on Sephadex LH-20 (methanol), 41 mg of compound 7 were isolated. Fractions Fr4+5 (480 mg) presented two separated components and were chromatographed over silica gel CC using a gradient of CHCl3–MeOH (100:0 to 70:30) as eluent to yield 8 subfractions (Fr(4+5).1 to Fr(4+5).8). Fr(4+5).5 (154 mg) was purified by CC on silica gel eluting with CHCl3– MeOH (100:0 to 80:20) and followed by filtration on Sephadex LH-20 CC using CHCl3– MeOH (80/20) as eluent to afford three compounds 3 (14 mg), 2 (5 mg) and 1 (4 mg). 4. Spectroscopic data l-O-palmitoyl-3-O-(6-sulfo-α-D-quinovopyranosyl)-glycerol (1) White amorphous powder; mp: 143; [α]D +47.1° (C = 0.035, MeOH); IR (KBr) υmax: 3400, 1730, 1170, 1150, 1060, 1035, 771 cm-1; 1H NMR (500 MHz, CD3OD) δH (ppm): 4.05 (1H, m, H-3a), 3.39 (1H, m, H-2b), 4.07 (1H, m, H-2a), 4.20 (1H, dd, J = 5.9, 6.5 Hz, H-3a), 4.10 (1H, m, H-3b), 2.37 (1H, t, J = 7.6 Hz, H-2′′), 1.61 (1H, m, H-3′′),1.20–1.40 (m, H-4”–H15”), 0.90 (3H, t, J = 6.7 Hz, H-16′′), 4.78 (1H, d, J = 3.8 Hz, H-1′), 4.48 (1H, m, H-2′), 3.37 (1H, t, J = 8.9 Hz, H-3′), 3.08 (1H, t, J = 6.3 Hz, H-4′), 4.07 (1H, d, J = 6.3 Hz, H-5′), 2.92 (1H, dd, J = 9.2, 8.2 Hz, H-6′b), 3.35 (1H, J = 1.5, 2.0 Hz, H-6′a); 13 C NMR (500 MHz, CD3OD) δC (ppm): 70.7 (C-1), 69.9 (C-2), 66.7 (C-3), 175.9 (C-1′′), 35.1 (C-2′′), 26.1 (C-3′′), 23.9–33.2 (C-4"–C-15"), 14.6 (C-16′′), 100.4 (C-1′), 73.8 (C-2′), 75.3 (C-3′), 75.1 (C-4′), 96.9 (C-5′), 54.9 (C-6′); ESI-MS (negative mode) m/z 555 [M-H]- (C25H48O11S). 3 1,2-di-O-palmitoyl-3-O-(6-sulfo-α-D-quinovopyranosyl)-glycerol (2) White amorphous powder; mp: 122; [α]D +48.0° (C = 0.15, CH3Cl:MeOH = 5:1); IR (KBr) υmax: 3420, 1736, 1121, 1028 cm-1; 1H NMR (500 MHz, CD3OD) δH (ppm): 4.10 (1H, dd, J = 6.4, 5.8 Hz, H1-a), 3.57(1H, dd, J = 5.2, 5.4 Hz, H-2b), 5.31 (1H, m, H-2), 4.50 (1H, dd, J = 7.1, 7.0 Hz, H3-a), 4.12 (1H, dd, J = 3.0, 3.5 Hz, H3-b), 4.76 (1H, d, J = 3.6 Hz, H-1'), 3.40 (1H, dd, J = 3.5, 4.2 Hz, H-2'), 3.63 (1H, dd, J = 8.9, 2.3 Hz, H-3"), 3.09 (1H, t, J = 9.2 Hz, H-4'), 4.07 (1H, dd, J = 1.6, 1.6 Hz, H-5'), 2.93 (1H, dd, J = 9.3, 9.6 Hz, H-6'b), 3.36 (1H, m, H-6'a), 2.37(1H, t, J = 7.6 Hz, H-2", H-2'"), 1.61 (1H, m, H-3", H-3'"), 1.20–1.40 (m, H-4"– H-15", H-4'"–H-15'"), 0.90 (6H, t, J = 6.7 Hz, H-16", H-16'"); 13C NMR (500 MHz, CD3OD) δC (ppm): 69.6 (C-1), 71.8 (C-2), 66.6 (C-3), 175.07 (C-1"), 175.2 (C-1'"), 35.0 (C-2", C-2'"), 26.1 (C-3", C-3'"), 30.4–31.0 (C-4"–C-15", C-4'"–C-15'"), 14.6 (C-16", C-16'"), 100.4 (C-1'), 73.8 (C-2'), 75.2 (C-3'), 75.0 (C-4'), 96.9 (C-5'), 54.9 (C-6'); ESI-MS (negative mode) m/z 793 [M-H]- (C41 H79O12S). Soyacerebroside I (3) White amorphous powder; mp: 114; [α]D +3.358° (C = 0.358, MeOH:CHCl3 = 3:2); IR (KBr) υmax: 3455, 2975, 1658, 1610, 1328, 1130, 1025 cm-1; 1 H NMR (500 MHz, CD3OD) δH (ppm): 0.90 (6H, t, J = 6.7 Hz, H-16', H-18 ), 1.20-1.35 (m, H-11–H-17, H-5'–H-15'), 1.42 (1H, m, H-4'), 1.58 (1H, ddd, J = 14.0, 8.4 Hz, H-3'a), 1.70 (1H, dq, J = 14.7 Hz, H-3'b), 1.97 (1H, m, H-10), 2.06 (1H, m, H-6), 2.07 (1H, m, H-7), 3.19 (1H, dd, J = 8.8, 7.7 Hz, H-2"), 3.28 (1H, m, H-4"), 3.31 (1H, m, H-5"), 3.36 (1H, t, J = 9.0 Hz, H-3"), 3.67 (1H, dd, J = 12.0, 5.1 Hz, H-6"a), 3.71 (1H, dd, J = 10.0, 3.5 Hz, H-1a), 3.81 (1H, dd, J = 12.0, 1.4 Hz, H-6"b), 3.98 (1H, ddd, J = 7.5, 5.0, 3.5 Hz, H-2), 4.00 (1H, dd, J = 8.0, 3.5 Hz, H-2'), 4.10 (1H, dd, J = 10.0, 5.5 Hz, H-1b), 4.12 (1H, m, H-3), 4.27 (1H, d, J = 7.7 Hz, H-1"), 5.42 (2H, m, H-8, H-9), 5.47 (1H, dd, J = 15.0, 7.0 Hz, H-4), 5.73 (1H, m, H-5). 13C NMR (500 MHz, CD3OD) δC (ppm): 14.3 (C-16' and C-18), 30.37–31.01 (m, C-11–C-16, C-6'–C-14'), 23.2 (C-15', C-17 and C-4'), 32.8 (C-10), 33.1 (C-7), 35.2 (C-3'), 53.9 (C-2), 62.1 (C-6"), 69.0 (C-1), 70.8 (C4"), 72.4 (C-3), 72.6 (C-2'), 74.2 (C-2"), 77.1 (C-3" and C-5"), 103.8 (C-1"), 129.8 (C-4), 130.0 (C-9), 131.6 (C-8), 134.2 (C-5), 177.2 (C-1'); ESI-MS (negative mode) m/z 712 [M-H], ESI-MS (positive mode) m/z 736 [M+Na]+ (C40 H75O9N). 4 8-oxo-pinoresinol (4) White powder ; mp: 110 °C; [α]D -3.5° (C = 0.20, MeOH); IR (KBr) υmax: 3430, 1753, 1631, 1600, 1501, 1230 and 1021 cm-1 ; 1H NMR (500MHz, CDCl3) δH (ppm): 3.27 (1H, m, H-1), 5.36 (1H, dd, J = 3.5, 3.8 Hz, H-2), 4.07 (1H, dd, J = 4.5, 4.5 Hz, H-4b), 4.36 (1H, dd, J = 6.6, 6.6 Hz, H-4a), 3.27 (1H, m, H-5), 5.34 (1H, dd, J =3.5, 3.5 Hz, H-6), 6.81 (1H, m, H-2'), 6.82 (1H, m, H-6'), 6.88 (1H, m, H-2"), 6.90 (1H, m, H-6"); 13C NMR (500 MHz, CDCl3) δC (ppm): 53.5 (C-1), 83.5 (C-2), 72.8 (C-4), 50.1 (C-5), 84.8 (C-6), 177.2 (C-8), 131.2 (C-1'), 107.9 (C-2'), 147.1 (C-3'), 146.2 (C-4'), 114.9 (C-5'), 118.6 (C-6'),132.4 (C-1"), 108.2 (C-2"), 146.9 (C-3"), 145.6 (C-4"), 114.6 (C-5"), 118.2 (C-6") and 55.6 (C-3', C-3"); ESI-MS (negative mode) m/z 371 [M-H]-, ESI-MS (positive mode) m/z 395 [M+Na]+ (C20H20O7). Quercetin (5) Yellow powder; mp: 316.3°C; UV (AlCl3 + MeOH) λmax : 272, 448 nm; IR (KBr) υmax: 3500, 1662, 1614, 1512 cm-1; 1H NMR (500MHz, CD3OD) δH (ppm): 6.20 (1H, d, J = 2.0 Hz, H-6), 6.40 (1H, d, J = 2.0 Hz, H-8), 7.75 (1H, d, J = 2.1 Hz, H-2'), 6.9 (1H, d, J = 8.5 Hz, H-5'), 7.65 (1H, dd, J = 8.5, 2.1 Hz, H-6’); 13 C NMR (500 MHz, CD3OD) δC (ppm): 148.2 (C-2), 137.2 (C-3), 177.5 (C-4), 162.6 (C-5), 99.4 (C-6), 165.7 (C-7), 94.6 (C-8), 158.4 (C-9), 104,7 (C-10), 124.3 (C-1'), 116.1 (C-2'), 146.3 (C-3'), 150.3 (C-4'), 116.2 (C-5'), 121.8 (C-6'); ESIMS (negative mode) m/z 303 [M-H]-, ESI-MS (positive mode) m/z 325 [M+Na]+ (C15H10O7). Apigenin (6) Yellow powder; mp : 347.5°C ; UV (AlCl3 + MeOH) λmax: 341, 271 nm; IR (KBr) υmax: 3340, 1656, 1610, 1508 cm-1; 1H NMR (500 MHz, CD3OD) δH (ppm): 6.21 (1H, s, H-6), 6.49 (1H, s, H-8),6.59 (1H, s, H-3), 6.90 (1H, d, J = 8.8 Hz, H-3'), 6.90 (1H, d, J = 8.8 Hz, H-5'), 7.40 (1H, d, J = 8.8 Hz, H-2'), 7.40 (1H, d, J = 8.8 Hz, H-6'); C NMR (500 MHz, CD3OD) δC 13 (ppm): 164,6 (C-2), 102.4(C-3), 182.4(C-4), 161.8(C-5), 98.7(C-6), 164.9 (C-7), 93.5 (C-8), 149.5 (C-9), 103.9 (C-10), 122.3 (C-1'), 127.5 (C-2'), 115.3 (C-3'), 158.0 (C-4'), 115.3 (C-5') 127.5 (C-6'); ESI-MS (negative mode) m/z 371 [M-H]- (C15H10O5). Isovitexin (7) Yellow powder; mp: 286; UV (MeOH) λmax: 334, 270, 302 nm; IR (KBr) υmax: 3400, 1650, 1610, 1520 cm-1 ; 1H NMR(500 MHz, CD3OD) δH (ppm): 7.85 (2H, d, J = 8.5 Hz, H-3', H5'), 6.94 (2H, d, J = 8.4 Hz, H-2', H-6'), 6.71 (1H, s, H-3), 6.42 (1H, s, H-8), 4.56 (1H, d, J = 5 9.8 Hz, H-1"); 13C NMR (500 MHz, CD3OD) δC (ppm): 163.3 (C-2), 102.6 (C-3), 181.7 (C4), 160.6 (C-5), 108.9 (C-6), 163.3 (C-7),93.7 (C-8), 156.3 (C-9), 102.9 (C-10), 121.0 (C-1'), 128.3 (C-2', C-6'), 70.5 (C-2), 116.0 (C-3', C-5'), 78.9 (C-3"), 161.3 (C-4), 70.2 (C-4"), 73.1 (C-1"), 81.3 (C-5"), 63.3 (C-6"); ESI-MS (positive mode) m/z 471 [M + K]+ , m/z 455 [M + Na]+ (C21H20O10). Stigmat-7-en-3-ol (8) White crystals; IR (KBr) υmax: 3400, 2988, 2860, 1614 cm-1 ; 1H NMR (500 MHz, CDCl3) δH (ppm): 1.82 (1H, m, H-1a), 1.09 (1H, m, H-1b), 1.77 (H1, m, H-2a) 1,39 (H, m, H-2b), 3.52 (1H, m, H-3), 1.70 (H1, m, H-4a), 1.22 (1H, m, H-4b), 1.40 (1H, m, H-5), 1.74 (1H, m, H6a), 1.22 (1H, m, H-6b), 5.08 (1H, m, H-7), 1.65 (1H, m, H-9), 1.47 (1H, m, H-11), 1.98 (1H, m, H-12a), 1.18 (1H, m, H-12b), 1.18 (1H, m, H-14), 1.50 (1H, m, H-15a), 1.38 (1H, m, H15b), 1,80 (1H, m, H-16a), 1.23 (1H, m, H-16b), 1.25 (1H, m, H-17), 0,47 (3H, s, H-18), 1.13 (3H, s, H-19), 1.95 (1H, m, H-20), 0.97 (3H, d, J = 6.6 Hz, H-21), 1.36 (1H, m, H-22a), 1.07 (1H, m, H-22b), 1.21 (1H, m, H-23), 1.60 (1H, m, H-24), 1.49 (1H, m, H-25), 0.75 (3H, d, J = 6.6 Hz, H-26), 0,75 (3H, d, J = 6.6 Hz, H-27), 1,42 (1H, m, H-28a), 1,18 (1H, m, H-28b), 0,74 (3H, t, J = 7.4 Hz, H-29); 13C NMR (500 MHz, CDCl3) δC (ppm): 37.4 (C-1), 31.7 (C-2), 71.3 (C-3), 38.2 (C-4), 40.4 (C-5), 29.9 (C-6), 117.7 (C-7), 139.8 (C-8), 49.6 (C-9), 34.2 (C10), 21.7 (C-11), 39.7 (C-12), 43.5 (C-13), 55.4 (C-14), 23.2 (C-15), 28.7 (C-16), 56.1(C-17), 12.3 (C-18), 13.3 (C-19), 41.1 (C-20), 18.9 (C-21), 33.9 (C-22), 26.0 (C-23), 45.9 (C-24), 32.1 (C-25), 21.6 (C-26), 19.2(C-27), 25.6 (C-28), 12.5 (C-29); EI-MS m/z 414 [M] +. (C29H50O). Spinasterol (9) White crystals; IR (KBr) υmax: 3400, 2988, 2860, 1614 cm-1; 1H NMR (500 MHz, CDCl3) δH (ppm): 1.82 (1H, m, H-1a), 1.09 (1H, m, H-1b), 1.77 (H1, m, H-2a) 1,39 (H, m, H-2b), 3.52 (1H, m, H-3), 1.70 (H1, m, H-4a), 1.22 (1H, m, H-4b), 1.40 (1H, m, H-5), 1.74 (1H, m, H-6a), 1.22 (1H, m, H-6b), 5.08 (1H, m, H-7), 1.65 (1H, m, H-9), 1.47 (1H, m, H-11), 1.98 (1H, m, H-12a), 1.18 (1H, m, H-12b), 1.18 (1H, m, H-14), 1.50 (1H, m, H-15a), 1.38 (1H, m, H-15b), 1,80 (1H, m, H-16a), 1.23 (1H, m, H-16b), 1.25 (1H, m, H-17), 0.47 (3H, s, H-18), 1.13 (3H, s, H-19), 1.95 (1H, m, H-20), 0.97 (3H, d, J = 6.6 Hz, H-21), 4.95 (1H, dd, J =15.1, 5.8Hz, H-22), 5.08 (1H, dd, J = 15.1, 8.5 Hz, H-23), 1.60 (1H, m, H-24), 1.49 (1H, m, H-25), 0.75 (3H, d, J =6.6 Hz, H-26), 0,75 (3H, d, J = 6.6 Hz, H-27), 1,42 (1H, m, H-28a), 1,18 (1H, m, 6 H-28b), 0,74 (3H, t, J =7.4 Hz, H-29); 13C NMR (500 MHz, CDCl3) δC (ppm): 37.4 (C-1), 31.7 (C-2), 71.3 (C-3), 38.2 (C-4), 40.4 (C-5), 29.9 (C-6), 117.7 (C-7), 139.8 (C-8), 49.6 (C9), 34.2 (C-10), 21.7 (C-11), 39.7 (C-12), 43.5 (C-13), 55.4 (C-14), 23.2 (C-15), 28.7 (C-16), 56.1(C-17), 12.3 (C-18), 13.3 (C-19), 41.1 (C-20), 21.3 (C-21), 138.4 (C-22), 192.6 (C-23), 51.5 (C-24), 32.1 (C-25), 21.6 (C-26), 19.2 (C-27), 25.6 (C-28), 12.5 (C-29); EI-MS m/z 412 . [M] + (C29H48O). β-Sitosterol (10) White powder; mp: 136.4°C; [α]D -30°(C = 0.8, CHCl3); IR (KBr) υmax : 3398, 2943, 2863, 1639 cm-1 ; 1H NMR (500 MHz, CDCl3) δH (ppm): 1.90 (1H, m, H-1a), 1.13 (1H, m, H-1b), 1.88 (H1, m, H-2a), 1,56 (H, m, H-2b), 3.58 (1H, m, H-3), 2.34 (H1, ddd, J = 13.0, 5.0, 2.0 Hz, H-4a), 2.30 (1H, t, J = 10 Hz, H-4b), 5.40 (1H, dd, J = 5.2, 2.3 Hz, H-6), 1.50 (1H, m, H7), 2.03 (1H, m, H-8), 0.98 (1H, m, H-9), 1.55 (1H, m, H-11a), 1.50 (1H, m, H-11b), 2.03 (1H, m, H-12a), 1.21 (1H, m, H-12b), 1.04 (1H, m, H-14), 1.63 (1H, m, H-15a), 1.11 (1H, m, H-15b), 1,89 (1H, m, H-16a), 1.30 (1H, m, H-16b), 1.16 (1H, m, H-17), 0,74 (3H, s, H-18), 1.06 (3H, s, H-19), 1.40 (1H, m, H-20), 0.95 (3H, d, J = 5.1 Hz, H-21), 1,34 (1H, m, H-22a), 1,04 (1H, m, H-22b), 1,19 (2H, m, H-23), 0.97 (1H, d, J = 6.9 Hz, H-24), 1.71 (1H, m, H-25), 0.88 (3H, d, J = 6.9 Hz, H-26), 0,87 (3H, d, J = 6.9 Hz, H-27), 1,31 (3H, m, H-28), 0,89 (3H, t, J = 12.0 Hz, H-29); 13C NMR (500 MHz, CDCl3) δC (ppm): 37.2 (C-1), 31.6 (C-2), 71.8 (C3), 42.3 (C-4), 140.7 (C-5) 121.7 (C-6), 31.9 (C-7), 31.8 (C-8), 50.1 (C-9), 36.5 (C-10), 21.1 (C-11), 39.7 (C-12), 42.3 (C-13), 56.0 (C-14), 24.3 (C-15), 28.2 (C-16), 56.0 (C-17), 11.8 (C18), 19.4 (C-19), 36.1 (C-20), 18.8 (C-21), 33.9 (C-22), 26,0 (C-23), 45.8 (C-24), 29.1 (C-25), 19.8 (C-26), 19.0 (C-27), 23.0 (C-28), 12.0 (C-29), ; ESI-MS (negative mode) m/z 413 [M-H]ESI-MS (positive mode) m/z 437 [M+Na] + (C29H50O). β-Sitosterol-3-O-glucoside (11) White powder; mp: 285.2 °C; [α]D -41.5° (C = 0.4, MeOH); IR (KBr) υmax : 3421, 2943, 1639 cm-1; 1H NMR (500 MHz, CD3OD) δH (ppm): 1.88 (1H, m, H-1a), 1.08 (1H, m, H-1b), 1.91 (H1, m, H-2a) 1,62 (H, m, H-2b), 3.59 (1H, m, H-3), 2.42 (H1, d, J = 10 Hz, H-4a), 2.28 (1H, t, J = 10 Hz, H-4b), 5.37 (1H, d, J = 5.0 Hz, H-6), 1.47 (1H, m, H-7), 1.97 (1H, m, H-8), 0.96 (1H, m, H-9), 1.45 (1H, m, H-11a), 1.02 (1H, m, H-11b), 2.01 (1H, m, H-12a), 1.17 (1H, m, H-12b), 1.03 (1H, m, H-14), 1.59 (1H, m, H-15a), 1.11 (1H, m, H-15b), 1,84 (1H, m , H-16a), 1.29 (1H, m, H-16b), 1.14 (1H, m, H-17), 0,70 (3H, s, H-18), 1.03 (3H, s, H-19), 1.37 (1H, 7 m, H-20), 0.95 (3H, d, J = 5.1 Hz, H-21), 1,34 (1H, m, H-22a), 1,04 (1H, m, H-22b), 1,19 (2H, m, H-23), 0.93 (1H, m, H-24), 1.68 (1H, m, H-25), 0.94 (3H, d, J = 5.3 Hz, H-26), 0,83 (3H, d, J = 6.9 Hz, H-27), 1,26 (3H, m, H-28), 0,85 (3H, t, J = 7.4 Hz, H-29), 4.41 (1H, d, J = 7.9 Hz, H-1'), 3,23 (1H, t, J = 7.9 Hz, H-2'), 3,42 (1H, t, J= 7.9 Hz, H-3'), 3,42 (1H, m, H4'), 3.29 (1H, m, H-5'), 3,84 (1H, d, J = 10.0 Hz, H-6a), 3,75 (1H, dd, J = 10.0, 5.0 Hz, H6'b); 13C NMR (500 MHz, CD3OD) δC (ppm): 37.6 (C-1), 29.9 (C-2), 79.5 (C-3), 39.0 (C-4), 140.7 (C-5) 122.4 (C-6), 32.3 (C-7) 32.3 (C-8), 50.6 (C-9), 37.1 (C-10), 21.2 (C-11), 40.8 (C12), 42.7 (C-13), 57.2 (C-14), 24.6 (C-15), 28.6 (C-16), 56.4 (C-17), 12.1 (C-18), 19.5 (C19), 36.5 (C-20), 18.9 (C-21), 34.3 (C-22), 26,4 (C-23), 46.2 (C-24) 29.5 (C-25), 19.9 (C-26), 19.2 (C-27), 23.4 (C-28), 12.3 (C-29), 101.5 (C-1'), 74.0 (C-2'), 76.9 (C-3'), 70,7 (C-4'), 76.3 (C-5'), 62,2 (C-6'); ESI-MS (negative mode) m/z 575 [M-H]-, ESI-MS (positive mode) m/z 599 [M+Na] + (C35H60O6). 5. Total phenol content The concentration of total phenols in each plant extract (PDBU and PDAC) was determined with Folin–Ciocalteu reagent following the colorimetric method adapted by (Li et al. 2007). 200 µL of diluted sample were added to 1 mL of 1:10 diluted Folin–Ciocalteu reagent. After 4 min, 800 µL of saturated sodium carbonate (75 %) were added. After 2 h of incubation at room temperature, the absorbance at 765 nm was measured. Measurements were carried out in triplicate and calculations were based on a calibration curve obtained with gallic acid (0– 200 µg/mL). The levels of total phenols were expressed as micrograms of gallic acid equivalents per milligrams of extract (µg GAE ‘gallic acid equivalent’ /mg of extract). 6. Antioxidant activity 6.1. DPPH radical-scavenging assay The anti-radical activity of EtOAc, n-butanol and fraction containing glucolipids of P. dichothomus was determined by DPPH according to the procedure described by (Mansouri et al., 2005) with some modifications. 25 µL of diluted samples were added to 975 µL DPPH solution (6 × 10-5 M) and vortexes. After incubation in the darkness at room temperature during 30 minutes, the absorbance was determined at 517 nm, using a spectrophotometer in a 10 mm quartz cuvette. Methanol was used to zero the spectrophotometer. For negative control, the absorbance of the DPPH radical without sample was measured. The experiments 8 were done in triplicate. Quercetin was used as reference compound. The free radical scavenging activity was calculated using the following equation: The IC50 value is the concentration of the sample that scavenges 50 % of the DPPH radical, The ratio [phenolic] (µg)/[DPPH] (µg) was plotted against the % of remaining DPPH to obtain the amount of sample necessary to decrease the initial DPPH concentration by 50 % (EC50). The antiradical efficiency (AE) is calculated as follows: AE % = 1/EC50 6.2.β-Carotene linoleic acid assay The antioxidant activity of the examined plant extracts was evaluated using a β-carotenelinoleate model system following the method described by (Kartal et al. 2007). A stock solution of β-carotene/linoleic acid was prepared as follows: 0.5 mg of β-carotene was dissolved in 1 mL of chloroform; the chloroform was subsequently evaporated using a vacuum evaporator. Then 25 µL of linoleic acid and 200 mg of Tween 40 were added, then, 100 ml of distilled water saturated with oxygen (30 min at 100 mL/min) were added with vigorous shaking. Aliquots (2.5 mL) of this reaction mixture were transferred to test tubes, and 350 µL portions of the extracts were added before incubating for 48 h at room temperature. The same procedure was repeated with BHT, and a blank containing only 350 µL of methanol. After the incubation period, the absorbance was measured at 490 nm. Antioxidant capacities of the samples were compared with those of BHT and the blank. The absorbance of each sample was measured in different time intervals (0, 1h, 2h, 4h, 6h, 24h, 26h, 28h, and 48h). After 48 h, relative antioxidant activities (RAA %) of the extracts were calculated from the equation given below: RAA % = ASample/ABHT × 100 Where ABHT is the absorbance of the positive control BHT and ASample is the absorbance of the extract. 9 7. Antibacterial activity assay The antibacterial activity of the extracts (PDBU and PDAC) was evaluated in vitro by using disc-diffusion method (Sacchetti et al. 2005). Negative control was prepared using the DMSO which is employed to dissolve the samples. Each extract was subjected to serial dilution technique by using dimethyl sulphoxide as a solvent to give: 500 mg/mL, 250 mg/mL, 125 mg/mL and 65 mg/mL solutions. These different concentrations were tested against one Gram positive bacteria (Staphylococcus aureus ATCC 25923) and two Gram negative bacteria (Pseudomonas aeruginosa ATCC 27853 and Escherichia coli ATCC 25922) and two bacterial strains isolated from patient samples: Klebsiella pneumoniae BLSE and Enterobacter sp BLSE. Bacterial strains were cultured in Mueller Hinton. Whatman sterile filter paper discs (6 mm diameter) were impregnated with 5 µL of each dilution of P. dichotomus extracts and placed on the inoculated agar. The plates were incubated at 37 °C for 24 h and the inhibition zone diameter of the tested bacteria was determined to the nearest mm. Each experiment was replicated for three times. Gentamicin and ampicillin was used as reference drug controls. 8. Statistical analysis The data were expressed as mean±SD. One-way ANOVA analysis of variance and NewmanKeuls Multiple Comparison test were carried out to determine significant differences (p<0.05) between the means. The analyses were made using GraphPad Prism (Version 5.0). References Li HB, Cheng KW, Wong CC, Fan KW, Chen F, Jiang Y. 2007. Evaluation of antioxidant capacity and total phenolic content of different fractions of selected microalgae. Food Chim. 102:771–776. Kartal N, Sokmen M, Tepe B, Daferera D, Polissiou M, Sokmen A. 2007. Investigation of the antioxidant properties of Ferula orientals L. using a suitable extraction procedure. Food Chem. 100:584–589. Mansouri A, Guendez E, Kokkalou E, kefalas P. 2005. Phenolic profile and antioxidant activity of the Algerian ripe date fruit (phonix dactylifera). Food Chem. 89:411-420. Sacchetti G, Maietti S, Muzzoli M, Scaglianti M, Mansredini S, Radice M, Bruni R. 2005. Comparative evaluation of 11 essential oils of different origin as functional antioxidants, antiradicals and antimicrobial in food. Food Chem. 91:621–632. 10 BHT 1.5 PDBU absorbance 490mm PDAC 7 Fr(4+5).5 1.0 MeOH 0.5 0.0 0 20 40 60 time (h) Figure S1. Absorbance change of β-carotene at 490 nm in the presence of Pteranthus dichotomus extracts, negative and positive control (MeOH, BHT). 150 100 50 BH T PD BU PD A C 7 (4 +5 ).5 M eO H Fr 0 Figure S2. Antioxidant activity percentage of P. dichotomus determined by β-carotene bleaching test and comparison with the reference (BHT). 11 Table S1. The concentration that scavenges 50 % of DPPH (IC50), antiradical efficiencies (AE) and efficient concentrations (EC50) determined by quercetin and P. dichotomus extracts. Extracts/compound IC 50 (µg mL-1) EC50 (µg/µg DPPH) AE 7 358.888±11.213 15.337±0.479 0.065±0.002 PDBU 375.514±21.194 16.047±0.903 0.062±0.003 PDAC 691.333±88.143 29.544±3.766 0.034±0.004 Fr(4+5).5 912.667±40.203 39.002±1.718 0.029±0.013 QUER 1.149±0.0004 0.491±0.0 15.274±0.0 * Data are represented as mean±SD (n = 3). Significant difference (p<0.05) Table S2. Antimicrobial activity of PDAC and PDBU extracts expressed as diameter of the inhibition zones. 500 250 125 65 500 PDAC 250 (mg /mL) 125 65 AMP(µg) 10 PDBU (mg /mL) GEN(µg) 10 Escherichia coli Pseudomonas aeruginosa Staphylococcus aureus Klebsiella pneumoniae BLSE Enterobacter sp BLSE 10±2.828 8±1.414 7±0.0 15 20 10.67±1.527 10±1.732 9.67±1.527 8.33±0.577 32 13.5±0.707 8±1.414 - 8.33±0.577 7±0.0 - 14 19 28 14 12 * Values are the average of three replicates. (-): No inhibition. * AMP: ampicillin; GEN: gentamicin. 12 Compound 1: l-O-palmitoyl-3-O-(6-sulfo-α-D-quinovopyranosyl)-glycerol (Diop et al. 2004). Mass spectrum ESI-MS of compound 1 (negative mode) 1 H-NMR spectrum of compound 1 (500 MHz, MD3OD) 13 C-NMR spectrum of compound 1 (500 MHz, CD3OD) 13 Compound 2: 1,2-di-O-palmitoyl-3-O-(6-sulfo-α-D-quinovopyranosyl)-glycerol (Plouguerné et al. 2013) Mass spectrum ESI-MS of compound 2 (negative mode) 1 H-NMR spectrum of compound 2 (500 MHz, MD3OD) 13 C-NMR spectrum of compound 2 (125 MHz, CD3OD) 14 Compound 3 : Soyacerebroside I (Voutquenne et al. 1999). Mass spectra ESI-MS of compound 3 (positive and negative modes) 1 H-NMR spectrum of compound 3 (500 MHz, CD3OD) 13 C-NMR spectrum of compound 3 (125 MHz, CD3OD) 15 Compound 4: 8-oxo-pinoresinol (Kui-Wu et al. 2012). Mass spectrum ESI-MS of compound 4 (positive and negative modes) 1 H-NMR spectrum of compound 4 (500 MHz, CDCl3) 13 C-NMR spectrum of compound 4 (125 MHz, CDCl3) 16 Compound 5: Quercetin (Choi et al. 2006). Mass spectrum ESI-MS of compound 5 (positive mode) 1 H-NMR spectrum of compound 5 (500 MHz, CD3OD) 13 C-NMR spectrum of compound 5 (125 MHz, CD3OD) 17 Compound 6: Apigenin (Benabdelaziz et al. 2014). Mass spectrum ESI-MS of compound 6 (positive mode) 1 H-NMR spectrum of compound 6 (500 MHz, CD3OD) 13 C-NMR spectrum of compound 6 (125 MHz, CD3OD) 18 Compound 7: Isovitixine (Peng et al. 2005), Mas Masss spectra ESI-MS of compound 7 (positive mode) 1 H-NMR spectrum of compound 7 (500 MHz, CD3OD) 1 3 C-NMR spectrum of compound (7) (125 MHz, CD3OD) 19 Compounds 8 and 9: a mixture of two phytosterols: Stigmat-7-en-3ol (Smith et al. 1975) and Spinasterol (Ragasa et al. 2005). Mass spectrum EI-MS of compound 8 and 9 1 H-NMR spectrum of compound 8 and 9 (500 MHz, CDCl3) 20 13 C-NMR spectrum of compound 8 and 9 (125 MHz, CDCl3) Compound 10 : β-Sitosterol (Haba et al. 2007). Mass spectra ESI-MS of compound 10 (positive and negative modes) 21 1 H-NMR spectrum of compound 10 (500 MHz, CDCl3) 13 C-NMR spectrum of compound 10 (125 MHz, CDCl3) 22 Compound 11: β-Sitosterol-3-O-glucoside (Burdi et al. 1991). Mass spectra ESI-MS of compound 11 (negative and positive modes) 1 H-NMR spectrum of compound 11 (500 MHz, CD3OD) 23 13 C-NMR spectrum of compound 11 (125 MHz, CD3OD) 24