Effects of Grape Cell Culture Extracts on Human Topoisomerase II Catalytic Activity and Characterization of Active Fractions

Abstract

Grape and its cell culture extracts are rich in flavonoids and stilbenes that are biologically active. The objective of this study was to evaluate possible inhibitory effects of grape (a Vitis hybrid Bailey Alicant A) cell culture extract and subfractions on human DNA topoisomerase II catalytic activity and to characterize constituents in the most potent fractions. At 5 μg/mL, grape cell crude extract and Toyopearl (TP) fractions 2−6 provided significantly greater inhibition of topoisomerase II catalytic activity than quercetin, a chemopreventive agent previously known as a topoisomerase catalytic inhibitor. The most potent topoisomerase II catalytic inhibitors from grape cell culture extracts in descending order of potency were TP fractions 4 and 6 (IC₅₀ = 0.28−0.29 μg/mL), TP-3 (IC₅₀ = 0.74 μg/mL), and crude extract (IC₅₀ = 1.02 μg/mL); each was significantly more potent than resveratrol (IC₅₀ = 18.0 μg/mL), another well-known chemopreventive topoisomerase II catalytic inhibitor. Using both high-performance liquid chromatography and liquid chromatography electrospray ionization mass spectrometry, constituents in TP-4 and TP-6 were characterized. These constituents included cyanidin-3,5-diglucoside, malvidin-3-acetylglucoside, peonidin-3-coumaryl-5-diglucoside, procyanidin B₁, procyanidin B₂, procyanidin B₅, procyanidin dimer digallate, procyanidin C₁, myricetin, and rutin, none of which have been previously characterized from grape cell cultures. The significant potency especially of TP-4 and TP-6 from grape cell cultures suggests that these fractions may have potential as chemopreventive agents. Keywords: Human topoisomerase II; catalytic inhibitor; antitopoisomerase II activity; Vitis; grape cell culture; chemopreventive agents