Background: Betulinic acid, a naturally abundant, plant derived, pentacyclic triterpenoid possesses anti- HIV, anti-malarial and anti-inflammatory properties and has recently emerged as a potent
anti-tumor compound. This study explores the mode of action of betulinic acid on eukaryotic topoisomerase I and identifies the major functional group responsible along with more potent derivatives.
Material/Methods: Topoisomerase I relaxation activity was electrophoretically measured by the decreased mobility of the relaxed monomers followed by ethidium bromide staining. DNA cleavage was studied by electrophoretic separation of the nicked monomers from the relaxed and supercoiled monomers in presence of ethidium bromide. In-vivo DNA cleavage was studied in blasted mouse splenocytes by the SDS-K+ trapping of 3H-DNA-topoisomerase I-camptothecin ternary complex.
Results: Betulinic acid exerts its inhibitory effect by preventing topoisomerase I-DNA interaction as a result of which the ‘cleavable complex’ is not formed. In consequence, it also acts as an antagonist
to camptothecin-mediated cleavage. A series of analogues modified at C-3, C-17 and C- 20 positions of betulinic acid were subsequently assayed for inhibition of topoisomerase I catalytic activity. Replacement of the 17-carboxylic group reduces the inhibitory effect and decarboxylation leads to the complete loss of inhibitory effect.
Conclusion: This study is the first detail report of betulinic acid as a very potent inhibitior of eukaryotic topoisomerase I and highlights the necessity of the carboxylic functional group. Dihydro betulinic acid is the most potent (IC50=0.5 µM) pentacyclic triterpenoid to inhibit eukaryotic topoisomerase I till date and can be exploited as a strong candidate for anti-tumor drug designing.

Keywords: Antineoplastic Agents, Phytogenic - chemistry, Antineoplastic Agents, Phytogenic - isolation & purification, Antineoplastic Agents, Phytogenic - metabolism, Camptothecin - metabolism, DNA - metabolism, DNA Topoisomerases, Type I, Eukaryotic - antagonists & inhibitors, DNA Topoisomerases, Type I, Eukaryotic - metabolism, DNA-Binding Proteins - metabolism, Enzyme Inhibitors - chemistry, Enzyme Inhibitors - isolation & purification, Enzyme Inhibitors - metabolism, Macromolecular Substances, Molecular Structure, Plant Extracts - chemistry, Plant Extracts - metabolism, Protein Binding, Triterpenes - chemistry, Triterpenes - isolation & purification, Triterpenes - metabolism