Electrochemical investigation of flavonolignans and study of their interactions with DNA in the presence of Cu(II)

Abstract

Flavonolignans, silybin and its derivatives (2,3-dehydrosilybin, 7-O-methylsilybin, 20-O-methylsilybin) and isosilybin were studied using ex situ (adsorptive transfer, AdT) cyclic and square wave voltammetry (SWV). The two oxidation steps were described for flavonolignans at potentials E_p1 + 0.5 V and E_p2 + 0.85 V depending on experimental conditions. An additional oxidation peak at E_p3 + 0.35 V was observed only for 2,3-dehydrosilybin. The anodic currents of flavonolignans are related to their electron transfer processes (oxidation of hydroxyl groups), which was supported by density functional theory (DFT) and B3P86 theory level. Our electrochemical results confirmed that 2,3-dehydrosilybin is a relatively strong antioxidant, which is strictly associated with oxidation at E_p3. The oxidation processes and antioxidant parameters of flavonolignans can be affected by transition metal complexation via hydroxyl groups. We found that silybin and 2,3-dehydrosilybin are able to chelate transition metals, especially Cu²⁺. The formation of silybin/Cu complexes was studied by AdT SWV and the observation was also confirmed using fluorescence spectroscopy. The electrochemical investigation of DNA interactions and damage caused in the presence of silybin/Cu complex and hydrogen peroxide is described. We present evidence that flavonolignans are involved not only in antioxidant abilities but also in the prooxidation effects under in vitro conditions.

Introduction

Flavonoids are a large group of naturally occurring polyphenolic compounds that are distributed in vascular plants. They are important constituents of human diets as fruit and vegetables and they are the pigments responsible for a variety of colors (from yellow to violet) [1], [2], [3]. A wide range of biological activities (including anti-inflammatory, anti-bacterial, hepatoprotective activities) are attributed to flavonoid antioxidant [4] and chelating [5], [6], [7] abilities. Flavonoids are benzo-γ-pyrone derivatives that can be divided into several groups according to their structural differences [3]. Here we focused on the flavonolignan silybin and its derivatives.

Silybin (Scheme 1), one of the main components of the silymarin extract from seeds of Silybum marianum (L.) Gaertn. (Asteraceae) [8], [9], is used in the prevention and treatment of liver diseases [10]. In addition to silybin, silymarin contains other congeners such as silydianin, silychristin, isosilybin, 2,3-dehydrosilybin, other flavonoids, e.g. taxifolin and quercetin, and about 30% unidentified polymeric phenolic compounds [11].

The electrochemical oxidation of flavonoids has been investigated using different electrochemical techniques including cyclic, differential pulse and square wave voltammetry (CV, DPV and SWV) [12], [13], [14], [15], [16], [17], [18]. CV was used to study the redox behavior and antioxidant properties of natural phenolics [18]. The antioxidant power of phenolic compounds is related to their oxidation potential(s) (E_p): a low potential (E_p < 200 mV) indicates an effective antioxidant, while increasing E_p usually suggests less effective antioxidant abilities. Oxidation of (+)-catechin, quercetin and rutin has been studied by CV, DPV and SWV [17]. Their oxidation was found to be linked to the catechol 3,4-dihydroxyl group and it is a reversible process depending on pH [16], [17]. All the above studies were based on in situ analyses using glassy carbon electrodes (GCE) [13], [14], [16], [17], [18], [19], [20], [21]. The antioxidant capacity of silybin was examined using CV at GCE [22], [23], [24], [25]. Of other carbon-based electrodes, screen-printed and carbon paste electrodes have been recently used as sensors for sensitive analysis of silybin and silymarin [26], [27].

Electrochemistry was used for studying the interactions of flavonoids with transition metals and the corresponding coordination complexes. Transition metals, Cu or Fe, can be complexed with the dihydroxyl group of the polyphenols. Such complexation leads to modulation of the antioxidant properties of flavonoids and production of free radicals causing DNA damage and inactivation of enzymes [2], [5], [7], [28], [29], [30]. The interactions of flavonoids with macromolecules at selected experimental conditions have also been studied by electrochemical methods. For example, the interactions of quercetin and rutin with DNA [20], [21] and hemoglobin [19] were investigated using voltammetric methods at GCE. In many papers the DNA damaging effects of polyphenols in presence of transition metals were described. There are no sufficient informations on the direct interactions (DNA binding) of flavonoids with DNA. It is known that some of polyphenol aglycones with the double bond between positions C2 and C3 and a hydroxyl group at position C3 (e.g. quercetin) can bind to DNA [31].

However, to date no detailed electrochemical study of the flavonolignans and use of ex situ voltammetry to study their metal coordination has been reported. The goals of this work were as follows: i) to study the oxidation and adsorption of silybin, 2,3-dehydrosilybin, two selectively methylated derivatives (7-O-methylsilybin, 20-O-methylsilybin), and isosilybin (Scheme 1) using ex situ voltammetry at a basal-plane pyrolytic graphite electrode (PGE), ii) to describe the redox properties (antioxidant capacity) and complexation capacity (chelating) of flavonolignans with metal ions, iii) to analyze the interactions of silybin, 2,3-dehydrosilybin and their metal complexes with DNA.