Flavonoids from Anemone tomentosa roots

Abstract

The genus Anemone (Ranunculaceae) comprises about 150 species, of which 52 are distributed in northern China. As triterpenoid saponins are the main effective components of the genus Anemone, it has extensive biological activities, such as antitumor, antibacterial, and insect antifeeding properties. Anemone tomentosa (Maxim.) Pei is a perennial herb that is commonly called “Da Huo Cao” in China [1]. Its roots have been used as traditional Chinese medicine for the treatment of dysentery, malaria, infantile malnutrition, carbuncles, etc. [2]. Previous phytochemical studies of A. tomentosa have led to the isolation of coumarins [3], triterpenoids, and sterols [4, 5], but there are no records of the flavonoid constituents. Herein we communicate the additional isolation and structural elucidation of eight known flavonoids 1–8, of which 1, 4, and 6 were isolated from plants of the genus Anemone for the first time. Flavonoids were found first in A. tomentosa. Roots of A. tomentosa were collected in September 2007 from Ziwuling Mountain areas of Gansu Province (China) and identified by Prof. Xiaoqiang Guo, Department of Life Science, Longdong University. Ground air-dried roots (5 kg) of A. tomentosa were extracted at room temperature with 80% aq. EtOH (2 25 L). The residue (448 g) was partitioned between H2O and petroleum ether, CHCl3, EtOAc, and n-BuOH, successively. The EtOAc fraction (103 g) was chromatographed over silica gel with gradient elution by n-hexane:EtOAc (25:1 15:1) and CHCl3:MeOH (10:1 2:1) and monitoring by TLC, in order to produce thirteen subfractions (E1-E13). Subfractions E2 and E8 were applied to a Sephadex LH-20 column with elution by MeOH, and recrystallized to give compounds 1 (30 mg) and 4 (12 mg). The n-BuOH extract (92 g) was subjected to repeated silica gel column chromatography by elution with MeOH:CHCl3 (3:10 2:1) and monitoring by TLC, in order to produce sixteen subfractions (B1-B16). Subfractions B2, B4, B7, B8, B10, and B12 were repeatedly subjected to silica gel or Sephadex LH-20 column chromatography and preparative TLC, and recrystallized to afford compounds 2 (45 mg), 3 (19 mg), 5 (8 mg), 6 (21 mg), 7 (32 mg), and 8 (13 mg). Compound 1, C15H10O7, yellow powder (CH3COCH3), mp 310–312 C, positive response to Mg–HCl, AlCl3, and FeCl3–K3Fe(CN)6 tests, negative response to Molisch test. UV spectrum (MeOH, max, nm): 256, 302, 376. IR (KBr, , cm– 1): 3409 (OH), 1663 (C=O), 1612, 1563, 1520 (Ar). Spectral data (EI-MS, PMR, and 13C NMR) for 1 agreed with those reported in the literature for quercetin [6]. Compound 2, C21H20O12, yellow needles (MeOH), mp 221–223 C, positive response to Mg–HCl and Molisch tests. UV spectrum (MeOH, max, nm): 207, 252, 301, 354. IR (KBr, , cm–1): 3398 (OH), 1656 (C=O), 1603, 1560, 1493 (Ar). MS (FAB, m/z): 465 [M + H]+, 303 [M + H – Glc]+. PMR (500 MHz, CD3OD, , ppm, J/Hz): 12.61 (1H, s, HO-5), 11.24 (1H, s, HO-7), 9.71 (1H, s, HO-4 ), 9.21 (1H, s, HO-3 ), 7.53 (1H, d, J = 2.0, H-2 ), 7.41 (1H, dd, J = 2.0, 8.4, H-6 ), 6.91 (1H, d, J = 8.4, H-5 ), 6.37 (1H, d, J = 2.0, H-8), 6.24 (1H, d, J = 2.0, H-6), 5.31 (1H, d, J = 7.5, H-1 ), 3.99 (1H, dd, J = 2.1, 12.0, H-6 a), 3.76 (1H, dd, J = 6.1, 12.0, H-6 b), 3.61–3.35 (4H, m, H-2 -5 ). 13C NMR spectra data agreed with those reported for quercetin-3-O-D-glucopyranoside [7]. Compound 3, C21H20O12, yellow crystals (MeOH), mp 245–247 C, positive response to Mg–HCl and Molisch tests. MS (FAB, m/z): 465 [M + H]+, 303 [M + H – Glc]+. PMR (500 MHz, CD3OD, , ppm, J/Hz): 12.50 (1H, s, HO-5), 7.48 (1H, d, J = 2.1, H-2 ), 7.40 (1H, dd, J = 2.2, 8.5, H-6 ), 6.90 (1H, d, J = 8.6, H-5 ), 6.56 (1H, d, J = 2.0, H-8), 6.30 (1H, d, J = 2.0, H-6), 5.34 (1H, d, J = 7.4, H-1 ), 3.85 (1H, dd, J = 2.0, 11.9, H-6 a), 3.76 (1H, dd, J = 6.1, 11.9, H-6 b), 3.65–3.31 (4H, m, H-2 -5 ). 13C NMR spectral data agreed with those reported in the literature for quercetin-7-O-D-glucopyranoside [8].