Notes Oleanane and Taraxerane Glycosides from the Roots of Gomphrena macrocephala Minpei Kuroda, † Taku Aoshima, † Mitsue Haraguchi, ‡ Maria Cla ´udia Marx Young, § Hiroshi Sakagami, ⊥ and Yoshihiro Mimaki* ,† Laboratory of Medicinal Pharmacognosy, Tokyo UniVersity of Pharmacy and Life Sciences, School of Pharmacy, 1432-1, Horinouchi, Hachioji, Tokyo 192-0392, Japan, Centro de Sanidade Animal, Instituto Biolo ´ gico, AVenida Conselheiro Rodrigues AVes, 1252, CEP 04014-002, Sa ˜ o Paulo, SP, Brazil, Sec ¸ a ˜ o de Fisiologia e Bioquı ´mica de Plantas, Instituto de Bota ˆ nica, AVenida Miguel Stefano, 3687, A Ä gua Funda, CEP 01061-970, Sa ˜ o Paulo, SP, Brazil, and Department of Dental Pharmacology, Meikai UniVersity School of Dentistry, 1-1, Keyaki-dai, Sakado, Saitama 350-0283, Japan ReceiVed April 21, 2006 Phytochemical screening of the roots of Gomphrena macrocephala, with particular attention to its triterpene glycoside constituents, has resulted in the isolation of two new oleanane glycosides (1 and 2) and a new taraxerane glycoside (3). The structures of 1-3 were determined as 11R,12R-epoxy-3-[(O--D-glucuronopyranosyl)oxy]olean-28,13-olide (1), 11R,12R-epoxy-3-[(O--D-galactopyranosyl-(1f3)-O-[-D-glucopyranosyl-(1f2)]--D-glucuronopyranosyl)- oxy]olean-28,13-olide (2), and 11R,12R-epoxy-3-[(O--D-glucuronopyranosyl)oxy]taraxer-14-en-28-oic acid -D- glucopyranosyl ester (3), respectively, on the basis of their spectroscopic data and the results of hydrolysis. The aglycones (1a and 3a) of 1-3 with an epoxy group showed cytotoxic activity against HSC-2 human oral squamous carcinoma cells. Gomphrena macrocephala St.-Hill. is a perennial herb belonging to the family Amaranthaceae and one of the well-known Brazilian medicinal plants. A decoction prepared from the roots of G. macrocephala has long been used as a tonic and stimulant in Brazil. 1 However, a literature survey showed that no systematic investiga- tions have been done on G. macrocephala roots. 2 In connection with our work on bioactive secondary metabolites from traditional medicines, a chemical investigation has been done on the n-BuOH- soluble fraction of the roots of G. macrocephala 80% EtOH extract. This has resulted in the isolation of two new oleanane glycosides (1 and 2) and a new taraxerane glycoside (3). This paper deals with the structure elucidation of the three new triterpene glycosides on the basis of their spectroscopic data and the results of hydrolysis. The cytotoxic activity of 1-3 and their aglycones 1a and 3a against HSC-2 human oral squamous carcinoma cells is also described. An 80% EtOH extract of the roots of G. macrocephala was suspended in H 2 O and then successively partitioned with EtOAc and n-BuOH. The n-BuOH-soluble portion was repeatedly subjected to column chromatography over silica gel, octadecylsilanized (ODS) silica gel, and Sephadex LH-20 to give 1 (98.6 mg), 2 (83.3 mg), and 3 (41.3 mg). Compound 1 was obtained as an amorphous solid. Its molecular formula was derived as C 36 H 54 O 10 by the HRESITOFMS data, showing an [M + H] + ion at m/z 647.3795, and 13 C NMR spectrum (36 carbon signals). The IR spectrum of 1 showed absorptions due to hydroxy groups at 3324 cm -1 and a five-membered lactone group at 1774 cm -1 . The 1 H NMR spectrum of 1 showed signals for seven tertiary methyl groups at δ 1.30, 1.26, 1.13, 0.98, 0.90, 0.88, and 0.81 (each s) and one anomeric proton at δ 5.03 (d, J ) 7.8 Hz). Enzymatic hydrolysis of 1 with -glucuronidase gave a known triterpenoid (1a), identified as 11R,12R-epoxy-3-hydroxyolean- 28,13-olide, 3 and D-glucuronic acid. D-Glucuronic acid, including its absolute configuration, was identified by direct HPLC analysis of the hydrolyzate, which was performed on an ion-exclusion column of sulfonated polystyrene with isocratic elution in 5 mM H 2 SO 4 , with detection carried out by using a combination of refractive index (RI) and optical rotation (OR) detectors. The linkage position of -D-glucuronic acid was shown to be at C-3 of the aglycone by detecting a correlation between the anomeric proton at δ 5.03 and the C-3 carbon at δ 88.8 in the HMBC spectrum. From the above evidence, the structure of 1 was elucidated as 11R,- 12R-epoxy-3-[(O--D-glucuronopyranosyl)oxy]olean-28,13- olide. Compound 2 had the molecular formula C 48 H 74 O 20 on the basis of the HRESITOFMS, exhibiting an [M + Na] + peak at m/z 993.4672, and 13 C NMR spectrum (48 carbon signals). The 1 H NMR spectrum of 2 showed signals for three anomeric protons at δ 5.73 (d, J ) 7.8 Hz), 5.31 (d, J ) 7.8 Hz), and 4.91 (d, J ) 7.6 Hz), along with seven tertiary methyl groups at δ 1.23, 1.21, 1.08, 1.05, 0.88, 0.81, and 0.78 (each s). Acid hydrolysis of 2 with 0.2 M HCl yielded D-glucuronic acid, D-glucose, and D-galactose as the carbohydrate moieties, whereas enzymatic hydrolysis of 2 with naringinase gave 1a. 3 The results of the hydrolyses and the 1 H and 13 C NMR data indicated that the carbohydrate moiety of 2 was composed of D-glucuronic acid, D-glucose, and D-galactose. When the 13 C NMR spectrum of 2 was compared with that of 1, the resonances due to C-2 and C-3 of the glucuronosyl moiety were displaced downfield by 3.2 and 8.9 ppm and were observed at δ 78.7 and 87.1, respectively. This suggests that the C-2 and C-3 hydroxy groups of the glucuronosyl moiety are the positions at which the additional D-glucose and D-galactose units are linked. In the HMBC spectrum, the anomeric proton resonances at δ 5.73 (H-1 of D-glucosyl), 5.31 (H-1 of D-galactosyl), and 4.91 (H-1 of D-glucuronosyl) exhibited correlations with the carbon signals at δ 78.7 (C-2 of glucuronic acid), 87.1 (C-3 of the glucuronic acid), and 89.4 (C-3 of aglycone), respectively. Accordingly, the structure of 2 was elucidated as 11R,12R-epoxy-3-[(O--D-galactopyrano- * To whom correspondence should be addressed. Tel: +81-42-676-4573. Fax: +81-42-676-4579. E-mail: mimakiy@ps.toyaku.ac.jp. † Tokyo University of Pharmacy and Life Science. ‡ Instituto Biolo ´gico. § Instituto de Bota ˆnica. ⊥ Meikai University School of Dentistry. 1606 J. Nat. Prod. 2006, 69, 1606-1610 10.1021/np068014r CCC: $33.50 © 2006 American Chemical Society and American Society of Pharmacognosy Published on Web 10/17/2006