Cyclic Triterpenoid Saponins from Campanula lactiflora

Turk J Chem 30 (2006) , 21 – 28. c TÜBİTAK Cyclic Triterpenoid Saponins from Campanula lactiflora Nurettin YAYLI1∗, Asu USTA, Ahmet YAŞAR1 , Osman ÜÇÜNCÜ1 , Canan GÜLEÇ1 , Mustafa KÜÇÜKISLAMOĞLU2 1 Department of Chemistry, Faculty of Arts and Sciences, Karadeniz Technical University, 61080, Trabzon-TURKEY e-mail: 2 Department of Chemistry, Faculty of Arts and Sciences, Sakarya University, 54100, Sakarya-TURKEY Received 22.09.2004 Two cyclic natural compounds, 3β-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl]-13α,14αepoxy-8α,12β,15-trihydroxy-(17E,21E)-17,21-campanuldien-60 (30)-olide, called lactifloroside A, 1, and 3β-O−[β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl]-13α,14α-epoxy-8α,12β-dihydroxy-(17E,21E)17,21-campanuldien-60 (30)-olide, called lactifloroside B, 2, were isolated for the first time from Campanula lactiflora and their structures deduced by high field 1D and 2D 400 MHz NMR, FT-IR, HPLC, GC-MS, (+/-) LC-MS/MS and (+) FAB-MS spectra. The aglycones of the 2 saponins were named 13α,14α-epoxy3β,8α,12β,15-tetrahydroxy-(17E,21E)-17,21-campanuldien-30-oic acid and 13α,14α-epoxy-3β,8α,12βtrihydroxy-(17E,21E)-17,21-campanuldien-30-oic acid, and designated as campanuloic acid and 15deoxycampanuloic acid, respectively. Key Words: Campanula lactiflora, lactifloroside A and B, campanuloic acid, 15-deoxycampanuloic acid, cyclic bisdemoside. Introduction The genus Campanula L. belongs to the family Campanulaceae 1 . One Campanula species, Campanula lactiflora Bieb., was naturalized in northern Turkey1 . Previous phytochemical studies on C. lactiflora have shown the presence of luteolin 7-β-D-glucopyranoside2 , luteolin3 and 40 -O-(p-hydroxybenzoyl)-isorhamnetin3,7-di-O-β-D-glucopyranoside, sitosterol β-D-glucoside, p-hydroxybenzoic acid and ethyl docosanoate4 , and triterpenes5 . In our continuing phytochemical investigation of the mixture of chloroform and methanol extracts of air-dried leaves of C. lactiflora, 2 new cyclic triterpene saponins (1, 2) were isolated. This paper reports the isolation and characterization of 2 new cyclic natural products, designated as lactifloroside A (1) and lactifloroside B (2), through spectral analyses. Some cyclic triterpene saponins were reported earlier and ∗ Corresponding author 21 Cyclic Triterpenoid Saponins from Campanula lactiflora, N. YAYLI, et al., named “cyclic bisdemosides” in the literature6,7 . Labdane-type structures are well known and characterized by spectral analyses8 . Experimental General and Instrumentation NMR spectra were recorded on a Varian NMR at 400 MHz instrument in C5 D5 N. (+) FABS were recorded on a ZabSpec MS instrument and (+/-) LC-MS/MS were carried out on a Micromass Quattro spectrometer. Infrared spectra were obtained with a Perkin-Elmer 1600 FT-IR (4000-400 cm−1 ) spectrophotometer. Optical rotations were measured with a Perkin-Elmer 241 polarimeter. Mps were obtained using a Kofler hot stage apparatus and are uncorrected. Flash CC was performed on silica gel (230-400 mesh) and reversedphase silica gel RP-18, and PTLC was performed with precoated reversed-phase silica gel RP-18 F254 S. Analytical HPLC was carried out on an Agilent 1100 series using a RI detector and the column was Zorbax Carbohydrate (4.6 mm ID x 250 mm, 5 µm) with CH3 CN/H2 O (75:25), flow 0.3 mL/min. GC-MS analysis was performed using an Agilent-5973 Network. A mass spectrometer with an ion trap detector in full scan under electron impact ionization (70 eV) was used. The chromatographic column for the analysis was a DB-1701P capillary column (25 m x 0.32 mm i.d., film thickness 0.25 µm). The carrier gas used was helium at a flow rate of 1 mL/min. The injection was performed in split mode (ratio 20:1) at 230 o C. Then a 1 µL crude fraction was injected and analyzed with the column held initially at 100 o C for 1 min and then increased to 300 o C with a 10 o C/min heating ramp and subsequently kept at 300 o C for 15 min. Plant Material. A whole C. lactiflora Bieb plant was collected in August 1996 at Arpalı, Trabzon Hills (∼2000 m), Turkey. A voucher specimen (Yaylı 9-1996 KTUK and KATO 12654-1996) has been deposited in the Department of Chemistry and Faculty of Forestry at Karadeniz Technical University, Turkey. The species was identified according to the Flora of Turkey1 . Extraction and Isolation. The extraction procedure was as described previously4 . Fraction CL7 (230 mg) was again purified by flash CC on silica gel (60 g, 230-400 mesh). The column was eluted with CHCl3 (60 mL) followed by a discontinuous gradient with CHCl3 -MeOH (2:0.5, 100 mL; 2:1, 50 mL, 1:1, 50 mL, 1:2, 50 mL), then with MeOH (50 mL) and finally with MeOH-H2 O (10:0.5, 50 mL) to give 33 fractions (∼ 8-10 mL each). After TLC analysis, fractions 7-23 (mixture), 24-28 (84 mg) (saponin mixture called CL72) and 29-33 (mixture) were combined. CL72 was rechecked by reverse-phase-18 TLC in a MeOH-H2 O (1.5:1) solvent system and found to be a mixture. It was rechromatographed by RP-18 (15 g) flash column chromatography (2 x 60 cm). The column was eluted by a discontinuous gradient with acetone-H2 O (1:1, 20 mL; 1:0.8, 20 mL, 1:0.6, 30 mL, 1:0.5, 30 mL, 1:0.2, 30 mL) and finally with acetone (30 mL) to give 32 fractions (∼ 5-6 mL each). After TLC analysis, fractions 20-21 (32 mg) were combined and rechromatographed by RP-18 PTLC (0.25 mm, 20 x 20 cm, 4 plates) using MeOH-H2 O (1.5:0.5) to give 5 bands; CL722C: 1, 14.3 mg, Rf = 0.40; CL722D: 2, 9 mg, Rf = 0.26; CL722A, CL722B and CL722E contained minor unidentified compounds, Rf = 0.63, Rf = 0.46, Rf = 0.16, respectively. 3β-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl]-13α,14α-epoxy-8α,12β, 15-trihydroxy-(17E ,21E )-17,21-campanuldien-60(30)-olide, (1): Amorphous powder, mp 143146 o C; [α]D +12.28o (MeOH; c 1.14 x 10−3 ); 1 H NMR (C5 D5 N, 400 MHz) and 13 C NMR (C5 D5 N, 100 22 Cyclic Triterpenoid Saponins from Campanula lactiflora, N. YAYLI, et al., MHz) δ (ppm) see Table 1; FAB-MS m/z (%); m/z = 835(48) [M+Na]+, 851(13) [M+K]+ , 550(12), 522(10), 393(43), 369(24), 322(75), 253(32), 193(55), 179(48) and 149(32). (+) LC-MS/MS m/z (%); m/z = 835(100) [M+Na]+ , 851(28) [M+K]+ ; (-) LC-MS/MS m/z (%); m/z = 811(45) [M-H]+ , 812(21) [M]+ , 491(25%), 254(48%), 126(100%); FT-IR cm−1 : 3427, 2942, 1699, 1646, 1451, 1388, 1074, 1032. 3β-O-[β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl]-13α,14α-epoxy-8α,12β-dihydroxy(17E ,21E )-17,21-campanuldien-60(30)-olide, (2): Amorphous powder, mp 158-162 o C; [α]D +9.61o (MeOH; c 5.2 x 10−4 ); 1 H NMR (C5 D5 N, 400 MHz) and 13 C NMR (C5 D5 N, 100 MHz) δ (ppm) see Table 1; FAB-MS m/z (%); m/z = 835(25) [M+Na]+ , 851(13) [M+K]+ , 552(3), 513(5), 391(13), 345(28), 253(32), 192(100) and 179(16); (+) LC-MS/MS m/z (%); m/z = 835(100) [M+Na]+, 851(62) [M+K]+ ; (-) LCMS/MS m/z (%); m/z = 811(55) [M-H]+ , 812(25) [M]+ , 325(28%), 182(62%), 126(100%); FT-IR cm−1 : 3435, 2940, 1695, 1645, 1456, 1386, 1275, 1124, 1075. Acid Hydrolysis of 1: Compound 1 (5 mg) was refluxe (...truncated)