Probing antioxidant activity of 2′-hydroxychalcones: Crystal and molecular structures, in vitro antiproliferative studies and in vivo effects on glucose regulation

Abstract

In order to better understand the antioxidant behavior of a series of polyphenolic 2′-hydroxychalcones, we describe the results of several chemical and biological studies, in vitro and in vivo. Single crystal X-ray methods elucidated their molecular structures and important intermolecular interactions such as H-bonding and molecular stacking in the crystal structures that contribute to our knowledge in explaining antioxidant activity. The results of experiments using the 1,1-diphenyl-2-dipicrylhydrazyl (DPPH) UV–vis spectroscopic method indicate that a hydroxyl group in position 5′ induces the highest antioxidant activity. Consequently, 2,2′,5′-trihydroxychalcone was selected for further study in vitro towards ROS scavenging in L-6 myoblasts and THP-1 human monocytes, where it shows an excellent antioxidant activity in a concentration range lower than that reported by most studies of related molecules. In addition, this chalcone shows a very selective activity: it inhibits the proliferation of leukemic cells, but it does not affect the normal L-6 myoblasts and human fibroblasts. In studying 2,2′,5′-trihydroxychalcone's effect on weight gain and serum glucose and insulin levels in Zucker fatty (fa⁻/fa⁻) rats we found that supplementing the diet with a 10 mg/kg dose of this chalcone (3 times weekly) blunted the increase in glucose that co-occurs with weight gain over the 6-week treatment period. It is concluded that 2,2′,5′-trihydroxychalcone has the potential to serve as a protective agent for some debilitating diseases.

Introduction

An increasing amount of research has focused on the potential health benefits of naturally occurring polyphenolic compounds in fruits and vegetables due to their high levels of biological activity and low toxicity. Among these compounds are the chalcones, a family of widely distributed biological precursors of flavonoids, and a number of studies have explored the biological activity of chalcone derivatives. Investigations have found applications for these molecules in alleviating oxidative tissue damage [1], [2], [3] and inflammatory diseases [4], [5], [6]; the effects appear to occur mainly through a modulation of NF-κB signaling, a pathway strongly linked to the inflammation process [7], but may also involve chalcone interactions with extracellular–regulated kinases, c-Jun kinases [8] and p38 MAPK [9]. Chalcones have been shown to be effective against leishmaniasis [10], [11] and in cardiovascular diseases [4], [12], [13], [14], and applications as analgesics, antimalarials, antipyretics, and antibacterials have also been reported (for a recent review on the subject see Ref. [15]). Chalcones may act as positive allosteric modulators of the GABA_A receptor [16] or inhibit the cell cycle through modulation of cyclins and cyclin-dependent kinases [17], [18], [19]. In line with this latter ability, their cytotoxic/antitumor capabilities, along with multiple suggestions for possible mechanisms, have been described [6], [17], [20], [21], [22], [23], [24]. For example, apoptosis induction, either by increasing the expression of proapoptotic pathways or by reducing the expression of the antiapoptotic ones has been reported. The chalcones are also active against tumor progression by inhibition of invasion and angiogenesis and consequent inhibition of metastasis. One of the most promising aspects of chalcone cancer treatments is that cancerous cells seem to be more sensitive to this cytotoxicity than normal cells [21]. Many studies have attempted to classify the chemical properties of chalcones and their derivatives; the collective conclusion is that, depending on their substituents and their molecular location, chalcones can function as potent antioxidants [1], [25]. In particular, hydroxylation, methoxylation and prenylation of chalcones can increase their antioxidant activity.

Some chalcones have been shown to have a potential benefit toward type 2 diabetes and insulin resistance, in analogy with resveratrol (3,5,4′-trihydroxystilbene). In fact, resveratrol is able to reduce the glycemic level, to protect the β cells and improve insulin action in diabetic patients [26]. The best documented effect of resveratrol is the anti-hyperglycemic effect both in obese rats and streptozotocin-treated rats [27], [28], [29] probably due to stimulation of glucose uptake [30]. Chalcone derivatives have been able to improve the diabetic condition particularly by inhibiting the aldose reductase pathway, the key enzyme of the polyol pathway and also through their antioxidant activity [31], [32]. Stilbenes are structurally related to chalcones, indeed, adding a carbonyl between the bridging double bond and an aromatic ring in stilbenes yields a chalcone framework. Chalcones consist of two aromatic rings interconnected by a highly electrophilic three carbon atom assembly (alpha, beta unsaturated carbonyl moiety). We were stimulated to study chalcones as we did recently with resveratrol and one of its metabolites [33]. Using single crystal X-ray diffraction we have determined the molecular structure of the chalcones shown in Scheme 1, and assessed their radical scavenging activity in vitro using UV–visible spectroscopy. We selected one chalcone, 2,2′,5′-trihydroxychalcone, for further study of its scavenging ability towards reactive oxygen species (ROS) in cells, monitored through fluorescence techniques, and also for in vivo studies of its physiological effects on circulating glucose and insulin in obese rats.