Flavonoids from Echinops echinatus

A new isoflavone glycoside, echinoside (7), together with 7-hydroxyisoflavone, kaempferol-4′-methylether, kaempferol-7-methylether, myrecetin-3-O-α-l-rhamnoside, kaempferol and kaempferol-3-O-α-l-rhamnoside, has been isolated from the whole plant of Echinops echinatus. The structure of echinoside has been established by chemical and spectral data. This is the first report of the occurrence of these flavonoids in E. echinatus.


Introduction
Echinops echinatus Roxb. (Family Asteraceae), which is distributed throughout India, is used in the Indian System of Medicine for the treatment of chronic fever, inflammations and various other diseases [1]. A number of terpenoids, flavonoids and other constituents have earlier been reported to be derived from this plant [2 -7]. The present study deals with the isolation of a new isoflavone glycoside designated echinoside (7) from the MeOH fraction of the whole plant of E. echinatus together with six known flavonoids.

Results and discussion
Chromatographic resolution of the methanolic extract of the whole plant of E. Echinatus yielded a glycoside, echinoside (7), mp 268 -2708C, C 27 H 30 O 12 (M þ þ H, 547, FAB-MS) which was recognised to be an isoflavone from its positive Shinoda test, UV absorption maxima at 255, 292, 312 (sh) nm and a singlet at d 8.35 (H-2) in its 1 H NMR spectrum.
The aglycone (8) showed in its 1 H NMR a characteristic singlet at d 8.36 (H-2) for isoflavonoid, eight aromatic protons and broad singlet at d 10.80 for the OH group identical to 7-hydroxyisoflavone [8]. This was confirmed by 13 C NMR and MS data and direct comparison with authentic sample.
The presence of two sugar units in glycoside 7 was evidenced from the appearance of two anomeric proton signals as doublets at d 4.80 and 4.90, respectively, for rhamnosyl and glucosyl units in 1 H NMR, which was further supported by two anomeric carbon signals at d 98.2 and 100.4 ppm in its 13 C NMR spectrum. 1 H NMR of 7 exhibited a doublet at d 1.12 (3H, d, J ¼ 6.5 Hz) for rhamnosyl CH 3 group together with other protons of one mole of glucose and one mole of rhamnose.

General experimental procedures
Melting points were determined on a Toshniwal apparatus and are uncorrected. Infrared spectra were recorded in KBr pellet on a Perkin-Elmer spectrophotometer model 720 and UV on a Carry-14 spectrophotometer using spectral MeOH. 1 H and 13 C NMR images were taken at 100, 300 and 400 MHz NMR on a Bruker HX-90 in CDC1 3 and DMSO-d 6

Plant material
The whole plant of Echinops echinatus was collected from Varanasi district, India and identified by Dr N.K. Dube, Department of Botany, Banaras Hindu University. A specimen sample is preserved in the department.   3.3.4 Methanolysis of permethylated product (9). Compound 9 was refluxed with 6% HCl in MeOH for 3 h. The MeOH was removed from the reaction mixture which was then extracted with CHCl 3 . The CHCl 3 extract on crystallisation from MeOH furnished 7-hydroxyisoflavone (8), mp 2158C, which was identified by spectral data and comparison with authentic sample (mmp, co-TLC). The sugar residues in the hydrolysate was identified as 3,4,6trimetthoxy-D-glucose (10) and 2,3,4-trimethoxy-L-rhamnose (11) by co-PC with authentic samples available in the laboratory.