Quadranosides I-V, New Triterpene Glucosides from the Seeds of Combretum quadrangulare I Ketut Adnyana, † Yasuhiro Tezuka, † Arjun H. Banskota, † Quanbo Xiong, † Kim Qui Tran, ‡ and Shigetoshi Kadota* ,† Institute of Natural Medicine, Toyama Medical and Pharmaceutical University, 2630-Sugitani, Toyama 930-0194, Japan, and National University Ho Chi Minh City, Ho Chi Minh City, Vietnam Received November 17, 1999 Five new triterpene glucosides, quadranosides I-V(1-5), have been isolated from a MeOH extract of the seeds of Combretum quadrangulare, together with 13 known compounds. The structures of compounds 1-5 were elucidated on the basis of spectroscopic analysis. Among the new triterpene glucosides, three compounds (1, 2, 5) showed significant hepatoprotective effects against D-galactosamine (D-GalN)/tumor necrosis factor-R (TNF-R)-induced cell death in primary cultured mouse hepatocytes. Combretum species (Combretaceae) are widely used in folk medicine for the treatment of hepatitis, malaria, respiratory infections, and cancer in different parts of Asia and Africa. 1 Combretum quadrangulare is a tree indigenous to eastern Asia that is commonly known as “Tram bau” in Vietnam. The seeds, leaves, and stem bark of the plant have been used in Vietnamese folk medicine as an anti- pyretic, antidysenteric, and antihepatitis agent. The seeds are administered orally together with ripe bananas as an anthelmintic for ascariasis and oxyuriasis. 2 Previous chemi- cal investigations have been undertaken only on the leaves and flowers of C. quadrangulare. 3-5 In our continuing study on hepatoprotective natural products, 6 it was found that a MeOH extract of the seeds of C. quadrangulare exhibited potent hepatoprotective activity on D-galactosamine (D- GalN)/tumor necrosis factor-R (TNF-R)-induced cell death in primary cultured mouse hepatocytes. Purification of the MeOH extract by passage over Si gel followed by prepara- tive TLC has afforded five new triterpene glucosides, quadranosides I-V(1-5, Chart 1), along with 13 known compounds (13-18). In this paper, we report the isolation and structure elucidation of these new triterpene glucosides together with their hepatoprotective activity. Results and Discussion The dried seeds of C. quadrangulare were extracted with MeOH, and evaporation under reduced pressure yielded a light brown MeOH extract, which showed a potent hepato- protective effect on D-GalN/TNF-R-induced cell death in primary cultured mouse hepatocytes (43.3% inhibition of cell death at 100 µg/mL). The MeOH extract was then subjected to column chromatography over Sephadex LH- 20, Cosmosil 75C 18 -OPN, and Si gel followed by preparative TLC, to afford five new triterpene glucosides (1-5), to- gether with 13 known compounds, 19R-hydroxyasiatic acid (6), 7 nigaichigoside F1 (7), 7 arjungenin (8), 8 arjunglucoside I(9), 8 pinfaensin (10), 9 2R,3,23-trihydroxyurs-12,19-dien- 28-oic acid -D-glucopyranosyl ester (11), 10 5-methoxy- (-)-isolariciresinol (12), 11 5-methoxy-9--xylopyranosyl- (-)-isolariciresinol (13), 11 (+)-gallocatechin (14), 12 (-)- epicatechin (15), 12 -sitosterol glucoside (16), gallic acid (17), and methyl gallate (18). Quadranoside I (1) was isolated as a colorless amorphous solid. The molecular ion peak at m/z 673.3923 in its HRFABMS suggested the molecular formula to be C 36 H 58 O 10 . The IR spectrum indicated the presence of hydroxyl (3400 cm -1 ), carbonyl (1720 cm -1 ), and olefinic (1640 cm -1 ) groups. The 1 H NMR spectrum of 1 displayed signals corresponding to six tertiary methyls (δ H 1.04, 1.44, 1.59,1.74, 1.76, 1.78), two exo-olefinic protons (δ H 4.79, 4.89), and oxygenated methine and methylene protons ascribable to a sugar unit. The 13 C NMR spectrum, on the other hand, showed 36 carbon signals including 6 primary, 10 secondary, 13 tertiary, and 7 quaternary carbons, suggesting 1 to be a triterpene monoglycoside. The 1 H and 13 C NMR data (Table 1), assigned to the aglycon moiety from its 1 H- 1 H COSY and FG-pulsed HMQC spectra, suggested that the aglycon is a lupane-type triterpene bearing three hydroxyls and a carboxyl group (C-28). 8 Two oxymethine protons at δ H 3.41 and 4.27 showed correlations in the 1 H- 1 H COSY spectrum, suggesting their vicinal arrangement. Furthermore, both of the protons had long- range correlations, in the FG-pulsed HMBC spectrum, with a quaternary carbon at δ C 40.8 assigned to C-4. This indicated that the position of the two hydroxyl groups should be at C-2 and C-3. Similarly, the position of the third hydroxyl group was determined to be at C-6 from the 1 H- 1 H COSY and the FG-pulsed HMBC spectra (Table 1). The sugar moiety of 1 was determined to be a glucose unit based on the coupling constants of each proton and the 13 C NMR chemical shifts (δ C 62.2, 71.2, 74.3, 78.8, 79.4, 95.5). The chemical shifts of the anomeric proton (δ H 6.40, d, J ) 7.9 Hz) and carbon (δ C 95.5) revealed that the glucose was attached to the carboxyl group (C-28). This was confirmed by a long-range correlation between the anomeric proton and the carboxyl carbon (δ C 176.5) in the FG-pulsed HMBC spectrum. Accordingly, the planar structure of quadrano- side I was determined as 1. The stereochemistry of 1 was determined by analysis of its coupling constants and ROESY data. The coupling constant (9.5 Hz) between H-3 and H-2 indicated the hydroxyl groups to have a 2R,3-orientation, which was further supported by the ROESY correlation between H-3 and H-5 (Figure 1). The broad singlet nature of H-6 suggested the hydroxyl group at C-6 should be -oriented, which was further supported by the intense cross-peak between H-6 and H 3 -23 in the ROESY spectrum. Thus, the * To whom correspondence should be addressed. Tel.: 81-76-434-7625. Fax: 81-76-434-5059. E-mail: kadota@ms.toyama-mpu.ac.jp. † Institute of Natural Medicine, Toyama Medical and Pharmaceutical University. ‡ National University Ho Chi Minh City. 496 J. Nat. Prod. 2000, 63, 496-500 10.1021/np990581+ CCC: $19.00 © 2000 American Chemical Society and American Society of Pharmacognosy Published on Web 03/07/2000