Bioactive Constituents from Asparagus cochinchinensis ⊥ Hong-Jie Zhang, † Kongmany Sydara, ‡ Ghee Teng Tan, † Cuiying Ma, † Bounhoong Southavong, ‡ D. Doel Soejarto, † John M. Pezzuto, †,§ and Harry H. S. Fong* ,† Program for Collaborative Research in the Pharmaceutical Sciences, Department of Medicinal Chemistry and Pharmacognosy (M/C877), College of Pharmacy, the University of Illinois at Chicago, 833 S. Wood Street, Chicago, Illinois 60612, and Traditional Medicine Research Center (TMRC), Ministry of Health, Vientiane, Laos, People’s Democratic Republic Received August 10, 2003 Bioassay-directed fractionation of the dried roots of Asparagus cochinchinensis led to the isolation of a new spirostanol saponin, asparacoside (1), two new C-27 spirosteroids, asparacosins A (2) and B (3), a new acetylenic derivative, 3′′-methoxyasparenydiol (4), and a new polyphenol, 3′-hydroxy-4′-methoxy-4′- dehydroxynyasol (6), as well as five known phenolic compounds, asparenydiol (5), nyasol (7), 3′′- methoxynyasol (8), 1,3-bis-di-p-hydroxyphenyl-4-penten-1-one (9), and trans-coniferyl alcohol (10). Compounds 1, 6, and 8 demonstrated moderate cytotoxicities in a panel comprised of KB, Col-2, LNCaP, Lu-1, and HUVEC cells, with IC 50 values ranging from 4 to 12 µg/mL. The structures were determined by spectroscopic and chemical methods. The dried roots of Asparagus cochinchinensis (Lourerio) Merrill (Asparagaceae) are used in Laos to treat chronic fever [Lao name of plant: Kheua Ya Nang Xang; voucher specimen K.Sydara037]. The plant also has a long history of use for treating fever, cough, kidney diseases, and benign breast tumors in China. 1 Phytochemically, they have been reported to contain monosaccharides, oligosaccharides, 2 polysaccharides, 3 furostanol oligosides, 4 and phenolic com- pounds. 5 As part of an International Cooperative Bio- diversity Group (ICBG) involving the collaboration of institutions in Vietnam, Laos, and the United States, 6 a MeOH extract prepared from the roots of A. cochinchinensis collected in Laos was shown initially to inhibit HIV-1 replication by 78% at 20 µg/mL, while being devoid of cytoxicity in the HOG.R5 cell line. Dried roots (5 kg) of this plant were, therefore, re-collected for bioassay-directed fractionation studies aimed at identifying novel anti-HIV constituents. However, as the anti-HIV bioassay-directed fractionation proceeded, cytotoxic fractions emerged. With each level of separation, the cytotoxicity of concentrated fractions increased, which led us to redirect our efforts toward the isolation of potential antitumor compounds. As a result, six cytotoxic compounds were isolated from the roots of A. cochinchinensis. The current paper describes the isolation, structure elucidation, and biological evaluation of the compounds isolated from this plant. Results and Discussion Separation of the CHCl 3 -soluble fraction of the MeOH extract of the dried roots of A. cochinchinensis utilizing parallel HIV-infectivity and toxicity assays in the HOG.R5 reporter cell line 7 led to the isolation of a new spirostanol saponin, asparacoside (1), two new C-27 spirosteroids, asparacosins A (2) and B (3), a new acetylenic derivative, 3′′-methoxyasparenydiol (4), and a new polyphenol, 3′- hydroxy-4′-methoxy-4′-dehydroxynyasol (6). In addition, the known compounds asparenydiol (5), 8 nyasol (7), 9 3′′- methoxynyasol (8), 10 1,3-bis-di-p-hydroxyphenyl-4-penten- 1-one (9), 11 and trans-coniferyl alcohol (10) were also obtained. 12 Asparacoside (1) was obtained as a white powder with a molecular formula of C 49 H 80 O 21 based on HRTOFMS and NMR (Tables 1-4) studies. Anomeric signals of four sugar units were observed in the 1 H and 13 C NMR spectra of 1 [δ H 5.38 (d, J ) 7.7 Hz), 5.30 (d, J ) 7.7 Hz), 5.01 (d, J ) 7.4 Hz), 4.74 (d, J ) 7.7 Hz) and δ C 105.7 (d), 105.3 (d), 105.2 (d), 101.4 (d)] (Tables 3 and 4). The aglycone of 1 was determined to be a spirostanol by comparison of its NMR data (Tables 1 and 2) with those of known spirostane- type steroids 13 and was identified as sarsasapogenin due to its NMR data being identical to those reported in the literature. 14,15 A partial acid hydrolysis of 1 afforded a mixture containing sarsasapogenin glycosides 1a-d, which were separated by preparative HPLC chromatography. Compound 1a contains a disaccharide group [δ H 5.40 (d, J ) 7.7 Hz), 4.96 (d, J ) 7.6 Hz) and δ C 106.0 (d), 102.0 (d)], which was determined to be a [-D-glucopyranosyl-(1f2)]- -D-glucopyranosyl unit according to 1D and 2D NMR spectral data (Tables 3 and 4) including HMBC. The disaccharide unit attached to the C-3 of the sarsasapogenin aglycone was determined by the presence of the HMBC correlation between the anomeric proton signal at δ H 4.96 and the signal at δ C 75.2 (d). Compound 1a was identified as 25(S)-5-spirostan-3-ol 3-O--D-glucopyranosyl-(1f2)- -D-glucopyranoside, a component of a mixture of spirostanol saponins, known as 25S-schidigerasaponin D5, which was originally reported as a 25S/25R mixture from the stems of Yucca schdigera. 15 Compounds 1b-d were elucidated as sarsasaponenin trisaccharides due to their characteristic sugar anomeric signals observed in the 1 H and 13 C NMR spectra (Tables 3 and 4). In addition to the glucopyranosyl- (1f2)]--D-glucopyranosyl unit, an additional sugar unit was revealed in the NMR spectra for both 1b and 1d. The additional sugar unit in both compounds was identified as R-L-arabinopyranosyl through analysis of the NMR spectral data. The R-L-arabinopyranosyl unit of 1b was connected to C-4′ of the inner -D-glucopyranosyl unit based on the presence of a HMBC correlation between the R-L-ara- binopyranosyl anomeric proton signal at δ H 4.98 and the C-4′ NMR signal at δ C 81.4 (d), which resulted in a ⊥ Dedicated to the late Dr. Monroe E. Wall and to Dr. Mansukh C. Wani of Research Triangle Institute for their pioneering work on bioactive natural products. * To whom correspondence should be addressed. Tel: (312) 996-5972. Fax: (312) 413-5894. E-mail: hfong@uic.edu. † Program for Collaborative Research in the Pharmaceutical Sciences. ‡ Traditional Medicine Research Center. § Current address: Schools of Pharmacy, Nursing, and Health Sciences, Purdue University, 575 Stadium Mall Dr., West Lafayette, IN 47907-2091. 10.1021/np030370b CCC: $27.50 © 2004 American Chemical Society and American Society of Pharmacognosy Published on Web 01/21/2004