DNA Binding Interactions and DNA Topoisomerase I Inhibition Activities of Crude Extracts from <i>Annona Squamosa</i> (L.) and <i>Annona Muricata</i> (L.) Fruits

Abstract

Some plants metabolites serve as antiprotozoal and antitumour by binding to nuclear enzyme; DNA Topo I affecting DNA function and cell survival. This study was aimed at screening DNA binding interactions and DNA Topo I inhibitory activity of crude extracts from fruits of Annona muricata (L) and Annona squamosa (L) which can form the basis of developing efficacious, safe and low cost antiprotozoal and antitumor agents. Aqueous, Methanolic, Ethyl acetate and Hexane extracts from fruits of two hypothesized antiprotozoal and antitumour plants; Annona muricata (L) and Annona squamosa (L) were screened for DNA-binding interaction and DNA Topo I inhibition. For DNA-methyl green test, 50 µL crude extracts were incubated with 200 μL DNA-methyl green in darkness at 25°C for 24 hours. Absorbance decrease at 650 nm using UV-vis spectrophotometer was calculated as a percentage of untreated DNA-methyl green value whereas with IC50 calculated by regression analysis. For DNA Topo I inhibitory activity, crude extracts were incubated in 10 µg/mL with 0.5 µg of supercoiled pBR322 DNA and 1U of DNA Topo I at 37°C for 2 hours, reaction terminated using stop buffer containing 3% SDS, 60 mM EDTA, 50% glycerol, 0.25% bromophenol blue. Products were determined by electrophoresis on 1% agarose gel in Tris-acetate-EDTA (TAE) running buffer at 65 V/cm for 2 hours. 24 extracts were studied, percentage decrease in absorbance were between 18.14±2.67 - 38.06±1.47 (Aqueous), 17.14±2.67 - 41.01±1.09% (Methanolic), 9.05±1.67 - 20.50±2.01% (Ethyl acetate) and 4.04±1.12 - 10.09±1.39% (Hexane)., IC50 values were between 50 μg/mL – 100 μg/mL (6), 100 μg/mL – 150 μg/mL (8), 150 μg/mL – 200 μg/mL (7) and <200 μg/mL (3). The activity against DNA Topo I mediated relaxation of supercoiled pBR322 DNA at 5 µM, 25 µM and 100 µM observed in 8 hits with percentage decrease in absorbance between 17.14±2.67 - 40.01±1.09% with IC50 between 62.97±3.37 μg/mL - 131.37±10.77 μg/mL. The extracts of A. muricata and A. squamosa showed DNA Topo I inhibitory activities by inhibiting the relaxation of supercoiled DNA pBR322. However, further studies need to be conducted on the purified fractions of aqueous and methanolic extracts.