In Vitro and in Vivo Anti-Leishmanial Activity of Triterpenoid Saponins Isolated from Maesa balansae and Some Chemical Derivatives Nils Germonprez, † Louis Maes, ‡ Luc Van Puyvelde, †,§ Mai Van Tri, § Duong Anh Tuan, § and Norbert De Kimpe* ,# Tibotec nv, Generaal De Wittelaan 11 bus 4, B-2800 Mechelen, Belgium, Department of Pharmaceutical Sciences, Antwerp University, Universiteitsplein, B-2610 Antwerp, Belgium, Institute of Chemistry, National Centre for Natural Sciences and Technology, 18 Hoang Quocc Viet Road, Cau Giay, Hanoi, Vietnam, and Department of Organic Chemistry, Faculty of Agricultural and Applied Biological Sciences, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium Received December 22, 2003 The methanolic extract from the leaves of the Vietnamese medicinal plant Maesa balansae showed potent in vitro and in vivo activity against the tropical protozoal parasite Leishmania infantum. Bioassay-guided purification of the extract led to the identification of six triterpenoid saponins, maesabalides I-VI (1-6), each having a strong and specific anti-leishmanial activity. Maesabalide III (3) and IV (4) were the most potent with IC 50 values against intracellular amastigotes of about 7 and 14 ng/mL. In comparison, the IC 50 value of sodium stibogluconate, the reference drug for treatment of leishmaniasis, is only 5.6 µg/mL. No cytotoxicity was present on a human fibroblast (MRC-5) cell line (CC 50 > 32µg/mL). In vivo evaluation in the BALB/C mouse model demonstrated that >90% reduction of liver amastigote burdens was obtained 1 week after a single subcutaneous dose at 0.2-0.4 mg/kg was administered. Several chemical derivatives of maesabalides I-VI were prepared in order to study the structure-activity relationship. Introduction In the course of a drug-screening project on medicinal plants for biologically active components, the Vietnam- ese medicinal plant Maesa balansae was examined and found to have strong anti-leishmanial potential. 1 Maesa balansae Mez. (Myrsinaceae), a shrub growing in the northern part of Vietnam, 2 is used in traditional medi- cine for the treatment of allergies, sprains, worm infections, skin ulcers, drunkenness, and headache. 3 The polar extract of the leaves exhibited potent and selective in vitro and in vivo activity against intracellular Leish- mania infantum amastigotes. 4 Bioassay-guided frac- tionation led to the isolation and characterization of six anti-leishmanial triterpene saponins designated as mae- sabalides I (1), II (2), III (3), IV (4), V (5), and VI (6) 5 (Chart 1). Leishmaniasis is a growing public health problem in many parts of the world with about 350 million people living in endemic areas and an annual incidence of about 2 million cases. 6 As for most other tropical diseases, inadequate resources are available to tackle this disease and treatment options are limited to pen- tavalent antimonials as first-line chemotherapeutics and to amphotericin and pentamidine as second-line chemotherapeutics. The recent approval of miltefosine as the first oral treatment of visceral leishmaniasis was a major step forward, but new drugs are still urgently needed and drug-screening initiatives should continue. 7 Natural products may offer unexpected chemical diver- sity for that purpose. Within the group of the saponin-containing plant species, anti-leishmanial activity has been reported only for the families Hedera, 8 Dracaena, 9 and Yucca. 10 Unfortunately, the in vitro screening methodologies mostly relied on promastigote models and/or the test concentrations were unrealistically high. In addition, conflicting results against intracellular amastigotes and no in vivo data in animal models were generated. This study evaluated the in vitro and in vivo anti- leishmanial activity and structure-activity relation- ships of maesabalides I-VI (1-6) and some semisyn- thetic derivatives (7-15) in an attempt to (a) establish a structure-activity relationship and (b) simplify and thereby increase the yield of the basic plant extract by derivatization. The fact that anti-leishmanial activity had been claimed for ursolic acid 11 at least opened some theoretical possibilities. Results and Discussion The dried leaves of M. balansae were extracted sequentially with dichloromethane and methanol. The residue obtained after evaporation of the methanol extract was partitioned between n-BuOH and water. The n-BuOH soluble fraction was evaporated to dryness. After the sample was stirred in acetone, the acetone- insoluble fraction (the “purified extract”) was repeatedly subjected to semipreparative reversed-phase HPLC in order to obtain the pure saponins, maesabalides I (1) * To whom correspondence should be addressed. Phone: +32 9 264 59 51. Fax: +32 9 264 62 43. E-mail: norbert.dekimpe@UGent.be. † Tibotec nv. ‡ Antwerp University. § National Centre for Natural Sciences and Technology. # Ghent University.