Quenching of cytotoxic reactive carbonyl species by black rice with giant embryo

Reactive carbonyl species (RCS) are cytotoxic molecules originating from lipid peroxidation and sugar oxidation [1]. Due to their involvement in the onset and propagation of different oxidative based diseases, such as atherosclerosis and diabetes related conditions, RCS are not only considered as biomarkers of oxidative stress, but also as promising drug targets. Recently small molecules acting as RCS sequestering agents have been reported and their promising pharmacological activity tested in different animal models [2]. Natural derivatives represent a very interesting source of potential RCS scavenging molecules but their investigation is limited, mainly by the lack of analytical methods able to screen and identify the bioactive compounds contained in complex matrices. To overcome this limit we recently set-up a robust and efficient method based on high resolution mass spectrometry to test the RCS scavenging activity of both small molecules and natural extracts [3]. In the present work such a method was applied to test the ability of water-soluble rice extracts to act as inhibitors of RCS-induced protein carbonylation. Ubiquitin, used as protein target, was incubated in vitro with different RCS, such as 4-hydroxy-trans-2-nonenal (HNE) and mixed with increasing concentrations of various rice extracts. After 24 hours of incubation, the reaction was stopped and the extent of ubiquitin carbonylation determined by HRMS. The dose-dependent inhibitory effect of the extracts was then calculated by measuring the extent of ubiquitin modification in the absence and the presence of the inhibitors. The different rice extracts inhibited the formation of RCS-induced protein modifications to different extents. Black rice sticky rice with giant embryo (IC50=~29.1 mg ml-1) varieties significantly inhibited protein modification by HNE and was more effective in respect to white rice (IC50=~151.0 mg ml-1). Identification of bioactive compounds was then carried out by an isotopic signature profile method using the characteristic isotopic ion cluster generated by the mixture of HNE:d5-HNE mixed at a 1:1 stoichiometric ratio. These findings suggest that certain bioactive components in water-soluble fractions of specific rice varieties effectively sequester RCS, and that such rice varieties can be valuable in dietary strategy for optimal health.