Antiproliferative Triterpene Saponins from Entada africana ⊥ Giuseppina Cioffi, † Fabrizio Dal Piaz, † Paolo De Caprariis, † Rokia Sanogo, ‡ Stefania Marzocco, † Giuseppina Autore, † and Nunziatina De Tommasi* ,† Dipartimento di Scienze Farmaceutiche, UniVersita ` di Salerno, Via Ponte Don Melillo, 84084 Fisciano (SA), Italy, and Departement de Medicine Traditionelle (DMT), INRSP, B.P. 1746 Bamako, Mali ReceiVed June 6, 2006 Nine new ester saponins (1-9) were isolated from the roots of Entada africana. Their structures were elucidated by 1D and 2D NMR experiments including 1D and 2D TOCSY, DQF-COSY, HSQC, and HMBC spectroscopy, as well as ESIMS analysis, and chemical methods. The aglycon moieties were found to be echinocystic acid for compounds 1, 2, 4-6, 8, and 9 and acacic acid for 3 and 7. All isolated compounds were tested for their antiproliferative activity against the J774.A1, HEK-293, and WEHI-164 cell lines. Moderate to high cytotoxic potency was found for almost all compounds tested. As a part of our investigation on Malian medicinal plants, we have conducted a phytochemical investigation on the roots of Entada africana Guill. and Perr. (Leguminosae), local name “Samane `re `”, a small tree growing in tropical areas of Africa. The plant is used in traditional medicine for various types of illnesses, and all parts of the tree are used. In Mali, various uses have been made of the anti-inflammatory, hepatoprotective, and wound-healing effects of E. africana. Decotions of the root or the bark are used to treat hepatitis and to wash wounds. The juice of the fresh root or the bark is also used for its hemostatic properties. Many other types of uses of E. africana are also reported in the literature in different parts of Africa. 1-3 Previous pharmacological studies on E. africana have shown anti- inflammatory, antiulcer, and wound-healing activities. 4-6 At the Departement de Medicine Traditionelle of Bamako, a traditional medicine, “Samane `re `”, is used to treat hepatitis. The clinical use of the “Improved Traditional Prescription”, which is prepared with the roots of E. africana, suggested its effectiveness in hepato- protection. 7 The antihepatotoxic properties of E. africana extracts were evaluated on CCl 4 -induced acute liver damage in rats. The results of this study showed that E. Africana does afford a protective action against CCl 4 -induced hepatocellular injury. 8 There is no report on the secondary metabolites of this plant to date. Previous phytochemical studies of other Entada species have reasulted in the occurrence of oleanolic acid, echinocystic acid, entagenic acid, and acacic acid glycosides. 9-11 In the present investigation on E. africana roots, we report the isolation and structural characterization of nine new triterpenoid ester saponins (1-9), having echinocystic acid and acacic acid as aglycons. Taking into account the fact that some triterpene saponins have been found to possess cytotoxic activity against various tumor cell lines, 12 all isolated compounds were tested for their anti- proliferative activity against the J774.A1, HEK-293, and WEHI- 164 cell lines, and the results are reported herein. Results and Discussion The methanol and chloroform-methanol extracts of roots of E. africana were subjected separately to Sephadex LH-20 column chromatography, followed by droplet countercurrent chromatog- raphy (DCCC), and then RP-HPLC, to afford nine triterpenoid saponins (compounds 1-9). Compound 1 was assigned a molecular formula of C 96 H 143 NO 43 , as determined by 13 C, 13 C DEPT NMR, and ESIMS. The HR/ ESIMS of 1 showed a signal at m/z 2020.9006, consistent with a [M + Na] + ion (theoretical m/z: 2020.8932; m/z difference 5 ppm). In the MS 2 spectrum, prominent fragments at m/z 1890 [M + Na - 130] + , 1758 [M + Na - (130 + 132)] + , and 1592 [M + Na - (130 + 132 + 166)] + were observed, due to consecutive losses of one cinnamoyl, one pentose, and one monoterpenoid residue, respectively. However the most abundant species was observed at m/z 1124 [M + Na - (130 + 766)] + and was produced by the loss of the whole esterified sugar chain. To obtain structural information on the molecule, a multistage MS n analysis was performed by fragmentation of the m/z 1124 parent ion generating a MS 3 spectrum, showing fragments at m/z 992 [M + Na - (130 + 766 + 132)] + , 962 [M + Na - (130 + 766 + 162)] + , and 860 [M + Na - (130 + 766 + 132 + 132)] + and suggesting the presence of at least one hexose unit and two pentose residues in the composition of the sugar chain glycosylated at C-3. 13 Finally, a fragment ion was detected at m/z 652 corresponding to the sodium-cationized ⊥ Dedicated to the memory of Prof. Arturo Leone, Universita ` di Salerno. * To whom correspondence should be addressed. Tel: +39-089-969754. Fax: +39-089-969602. E-mail: detommasi@unisa.it. † Universita ` di Salerno. ‡ INRSP, Bamako, Mali. 1323 J. Nat. Prod. 2006, 69, 1323-1329 10.1021/np060257w CCC: $33.50 © 2006 American Chemical Society and American Society of Pharmacognosy Published on Web 09/08/2006