Chemical Constituents of Linaria aucheri

Turk J Chem 28 (2004) , 133 – 139. c TÜBİTAK Chemical Constituents of Linaria aucheri Dilek ERCİL∗, M. Koray SAKAR Hacettepe University, Faculty of Pharmacy, Department of Pharmacognosy, 06100 Ankara, TURKEY e-mail: Esther DEL OLMO, Arturo SAN FELICIANO University of Salamanca, Faculty of Pharmacy, Department of Organic and Pharmaceutical Chemistry, 37007 Salamanca, SPAIN Received 10.06.2003 From the petroleum ether and methanol extracts of the aerial parts of Linaria aucheri (Scrophulariaceae) 6 known compounds: β-amyrin (1), ergost-7-en-3β-ol (2), stigmasta-5,22-E-dien-3β-ol (3), stigmast-5-en, 24S-3β-ol (4), antirrinoside (5) and linariin (6), were isolated and identified. The structures of these compounds have been elucidated by spectroscopic methods (UV, IR, 1 H NMR, 13 C NMR, DEPT-135 and GC-MS). Key Words: Linaria aucheri, Scrophulariaceae, lypophylic compounds, β-amyrin, antirrinoside, linariin. Introduction The genus Linaria (Scrophulariaceae) is represented by 20 species in the flora of Turkey1 . Several species have been used in traditional medicine as tonics, antiscorbutics, laxatives, antidiabetics and diuretics, as well as for the treatment of wounds, hemorrhoids and vascular disorders2,3 . Previous studies of Linaria species have shown the presence of flavonoids and their glycosides, as well as ionol glucosides, iridoids, alkaloids, diterpenoids and phenylethanoids3−12 . In the present paper, we report the initial isolation and identification of some known lypophylic compounds, together with an iridoid and flavon glycosides, from the aerial parts of Linaria aucheri petroleum ether and methanol extracts. Experimental General Experimental Procedures: UV spectra were obtained in MeOH on a Shimadzu spectrophotometer UV 160A. IR spectra were recorded on a Perkin Elmer spectrophotometer FTIR 1720X. The 1 H and 13 C spectra were obtained on a Bruker WP 200 SY spectrometer at 200 and 50.29 MHz, respectively. Silica gel 60 (0.063-0.200 mm, 7734) for column chromatography was purchased from Merck. Vacuum-liquid ∗ Corresponding author 133 Chemical Constituents of Linaria aucheri, D. ERCİL, et al., chromatography (VLC) was performed on a glass column (2.2 x 15 cm) filled with RP-18 (20-45 µm, LiChroprep) material. GC-MS apparatus: Hewlett-Packard 5890 Series II. Ion Sourced: 70 eV electron-impact ion source. Column used: SPB-1, methylsilicone, 12 m x 0.2 mm internal diameter, 0.33 µm width wall. Initial temperature: 100 o C, 5 o C increase per min. Final temperature: 300 o C for 5 min. Carrier gas: He. Plant Material: Linaria aucheri Boiss. (Scrophulariaceae) was collected from Baykuşboğazı, Çankırı (Northeast Anatolia) in July 1997. A voucher specimen has been deposited in the Herbarium of the Faculty of Pharmacy, Hacettepe University (HUEF 97-020). Extraction and Isolation: The air-dried and powdered aerial parts of L. aucheri (950 g) were ◦ extracted successively with petroleum ether in a mechanical stirrer (40-60 C) (3 x 2.5 l) and methanol (2 x 1.5 l). The petroleum ether extracts were evaporated to dryness in vacuo (10 g) and then defatted with acetone (6 x 100 mL). The crude extract (4.5 g) was subjected to the Si gel column (3 x 40 cm) with a gradient elution of 100% hexane to 25% ethyl acetate (1 l; 20 mL, each), to give 2 distinctive fractions (AI and AII). Fraction AI (Fr. 27-32, 338 mg) was purified by preparative TLC (5554 Merck) with a mixture of cyclohexane-ethyl acetate (8:2) to yield 1 (17 mg). Fraction AII (Fr. 34-48, 80 mg) is a mixture of compounds 2–4, with a ratio of 1:1:2, respectively. The methanolic extract (8 g) was subjected to RP-18 [20-45 µm, LiChroprep C-18 (Merck)] VLC with a gradient elution of methanol-water (10% to 90% MeOH) mixture. This yielded 5 main fractions (Frs. BI-BV, each of 100 mL). Fraction BII (1300 mg) yielded an iridoid (5), whereas fraction BV (110 mg) yielded a flavonoid (6). Acid Hydrolysis of 5 and 6: Compound 5 was applied to a TLC plate (Silica gel 60 F254 , 0.2 mm, Merck) and the plate was treated with concentrated HCl vapor in a closed tank for 1 h. After the evaporation of the concentrated HCl, authentic sugar samples were applied to the TLC plate, and the plate was developed in a EtOAc-MeOH-conc.HOAc-H2 O (60:15:15:10, v/v) solvent system. Spots were visualized ◦ by spraying with a Thymol-EtOH-conc.H2 SO4 (0.5 g:95 mL:5 mL) reagent and heated at 110 C for 5 min. The same procedure was used for compound 6, though this was treated for 2 h with concentrated HCl vapor. The sugars were identified as glucose for compound 5, and glucose and rhamnose for compound 6. Results and Discussion β-Amyrin (1): UV λmax (MeOH): 240.2 nm; IR γmax (KBr) cm−1 : 3397, 2932, 1645, 1465, 1379, 1029, 900; 1 H NMR (200 MHz, CDCl3 ): δ 5.12 (m, H-12), 3.23 (m, H-3), 1.13, 0.99, 0.97, 0.94, 0.87 (x 2), 0.83, 0.79 (CH3 ). 13 C NMR (50.29 MHz, CDCl3 ): δ 38.14 (C-1), 27.51 (C-2), 79.12 (C-3), 38.88 (C-4), 55.27 (C-5), 18.44 (C-6), 33.03 (C-7), 38.82 (C-8), 47.81 (C-9), 37.00 (C-10), 23.48 (C-11), 121.82 (C-12), 145.28 (C-13), 42.18 (C-14), 26.12 (C-15), 27.37 (C-16), 32.04 (C-17), 47.22 (C-18), 46.93 (C-19), 31.34 (C-20), 34.83 (C-21), 37.26 (C-22), 28.22 (C-23), 15.47 (C-24), 15.72 (C-25), 16.97 (C-26), 25.56 (C-27), 28.44 (C28), 33.44 (C-29), 23.48 (C-30); EI-MS: m/z 426 (M+ , 0.6%), 411 (0.1%), 218 (100%), 272 (0.1%), 189 (30%), 135 (34%), 95 (48%). Ergost-7-en-3β-ol (2): EI-MS: m/z 400 (M+ , 100%), 382 (43%), 367 (30%), 273 (32%), 255 (36%), 161 (48%), 147 (40%), 107 (77%), 43 (84%). Stigmasta-5,22E -dien-3β-ol (3): EI-MS: m/z 412 (M+ , 66%), 369 (11%), 300 (36%), 271 (55%), 134 Chemical Constituents of Linaria aucheri, D. ERCİL, et al., 255 (56%), 159 (60%), 147 (43%), 133 (58%), 97 (54%), 83 (100%), 55 (98%). Stigmast-5-en, 24S -3β-ol (4): EI-MS: m/z 414 (M+ , 98%), 381 (26%), 329 (52%), 303 (50%), 273 (33%), 213 (54%), 145 (76%), 119 (61%), 107 (88%), 95 (81%), 43 (100%). Antirrinoside (5): UV λmax (MeOH) nm: 212, 232 sh; IR γmax (KBr) cm−1 : 3392 (OH), 2922 (=C-H), 1657 (C=C), 1402, 1231, 1014, 960, 894, 860; 1 H and 13 C NMR (see Table 1). Table 1. 1 H and 13 C NMR spectroscopic data of 5 (δ ppm, CD3 OD). C/H atom Aglycone 1 3 4 5 6 7 8 9 10 Glucose 10 20 30 40 50 60 a 60 b δH δC 5.31 (d, J= 7.3 Hz) 6.30 (d, J = 6.4 Hz) 4.80 (d, J = 6.4 Hz) 3.88 (d, J = 1.6 Hz) 3.31 (d, J = 1.6 Hz) 2.30 (br.d, J = 7.3 Hz) 1.38 (3H,s) 95.0 142.9 107.6 74.5 78.1 66.2 64.1 53.0 17.6 4.59 (d, J = 7.3 Hz) 3.09-3.26* 3.09-3.26* 3.09-3.26* 3.09-3.26* 3.83 (dd, J = 1.8/11.7 Hz) 3.52 (dd, J = 6.2/11.7 Hz) 99.5 74.5 78.3 71.6 77.5 62.8 *Signal pattern unclear due to overlapping Linariin (6): UV λmax (MeOH) nm: , 230sh, 277, 328; (NaOMe) nm: 294.5; (AlCl3 ) nm: 230.5sh, 288.5, 300, 355; (AlCl3 +HCl) nm: 230.5sh, 289.5, 299, 353; (NaOAc) nm: 276, 328; (NaOAc+H3 BO3 ) nm: 276.5, 328.5; IR γmax (KBr) cm−1 : 3402, 2916, 1723, 1688, 1610, 1583, 1514, 1490, 1429, 1301, 1189, 835. 1 H and 13 C NMR (see Table 2). The 1 H NMR (...truncated)