Inhibition of Oxidative DNA Damage in the Presence of Spinach Extract

Abstract

Oxidative damage can induce DNA protein cross‐linking. The present study aimed to evaluate whether aqueous extracts of spinach can inhibit oxidative cross‐linking between DNA and protein. In the flash‐quench method, the intercalator Ru(phen)2dppz2+ (phen = phenanthroline, dppz = dipyridophenazine) is excited with blue light, releasing an electron to the quencher, Co(NH3)5Cl2+. The oxidized intercalator then extracts an electron from guanine and the guanine radical reacts to produce DNA‐protein cross‐links. Samples containing Co(NH3)5Cl2+, histone, Ru(phen)2dppz2+, and DNA were subjected to flash quench treatment in the absence or presence of a spinach extract. Cross‐linking was then detected in two ways—chloroform extraction assay and gel shift assays. With calf thymus DNA, the chloroform extraction assay was used, and this UV spectroscopy method showed that the spinach extract significantly decreased the loss of the free DNA absorbance due to crosslinking visible in a spectra and a plot of % crosslinked vs time. In gel shift assays, pUC19 DNA subjected to the flash quench technique decreased greatly in mobility, with a considerable amount of cross‐linked material visible within the wells. In the presence of an aqueous extract of spinach, cross‐linking is strongly inhibited and the DNA runs as a single band. This inhibition is maintained out to significant dilutions of the green extract. More recently, we have examined kaempferol, one antioxidant found in spinach, and have observed protection of DNA via a nicking assay. A single component of spinach would not be a universal protector against oxidative stress‐induced disease. Lipophilic components are also abundant, which is why two of them, vitamin K and β‐Carotene, are currently being examined.