1. Introduction
Flavonoids belong to a large group of polyphenolic compounds with the structure of benzo-γ-piron and are commonly found in plants. They are hydroxylated phenolic substances and are known to be synthesized by plants in response to microbial infection [
1]. Many flavonoid structures display a variety of biochemical properties, including estrogenic, antioxidant, antiviral, antibacterial, antiobesity, and anticancer activities. The diverse pharmacological activities of flavonoids have drawn considerable attention for their use in personal health care [
2]. With the publicity given to the beneficial effects, the consumption of dietary supplements containing flavonoids has increased significantly. It has been pointed out that the excess use of these compounds could have drastic effects, as high concentrations of flavonoids may act as mutagens, pro-oxidants and inhibitors of hormone metabolizing enzymes. Their chemical nature depends on their structural class, degree of hydroxylation and polymerization or other substitutions and conjugations. It affects their bioavailability and pharmacological action. Flavanone is the precursor to all flavonoid structures. Flavanones, which fall under a class of flavonoids, have important antioxidant properties [
3,
4] and interesting biological activities [
5,
6]. It has been indicated that flavanones with none or single OH group has anti-proliferation potential on colorectal carcinoma cells and mouse fibroblast NIH3T3 cells [
7,
8]. 6-Hydroxyflavanone is one of the members of above class of flavanoids. It is a monohydroxyflavanone which has hydroxy group at its 6th position. It has been reported [
9] that flavanone with the only hydroxylation at C6 has a significant cytotoxic effect in human leukemia HL-60 cells accompanied by the occurrence of apoptotic bodies, and hypodiploid cells, characteristics of apoptosis. In turn 2′-hydroxyflavanone, a nontoxic natural flavonoid has exhibited pleiotropic anticancer effects in many cancer types, including prostate and breast cancers [
10,
11]. On the other side, it has been found that 7-hydroxyflavanone has weak effect on inhibiting MCF-7 cell proliferation [
12] and low cytotoxic effect on acute lymphoblastic leukemia (ALL) and chronic myeloid leukemia (CML) cell lines [
13].
The above mentioned interesting pharmacological potential of flavanone and its monohydroxy derivatives prompted us to investigate these chemical compounds. Structure-related biological activities of flavanones are still considered unexplained. Since they exhibit various medicinal activities, it is intriguing to enter deeper into their chemical structures, electronic transitions or interactions with some biomolecules in order to find properties that allow us to better understand their effects. Such a knowledge will create the possibility of more conscious and purposeful use of these compounds both in medicine and in dietary supplements. Little information is available on biological activity of flavanone and its monohydroxy derivatives in relation to their physicochemical properties such as spectral profiles, existence of protonated/deprotonated species under pH changes or interaction with Calf Thymus DNA (CT DNA). Studies in solution are indispensable, because biologically active substances act in the cell compartments with different water contents, and therefore, the information about the acid–base properties give an idea of which chemical forms e.g., protonated/deprotonated of biomolecule may be involved in biochemical processes in the cells. We devoted this work to research aimed at demonstrating differences in the physicochemical properties of the four flavanones and linking them to their biological activity.
Recently, computational simulations are more and more frequently applied in the studies on electronic structures and properties of biologically important molecules. Theoretical studies may provide very useful and, in many cases unavailable experimentally, information about the structure and properties of biomolecules. Among various theoretical methods, density functional theory (DFT) approach has been extensively used in the prediction of structure-related properties of flavanones and structurally similar compounds [
14,
15,
16]. In particular it is possible to conclude about antioxidant activity of flavanones based on the analysis of the DFT predicted frontier orbitals and the DFT calculated quantum chemical descriptors [
14].
UV–Vis spectra of medium-size molecules such as flavanones and structurally related compounds are usually well predicted using the time-dependent TD DFT formalism [
14,
17,
18]. Within the framework of the TD DFT theory, the time-dependent oscillating electric field is incorporated in the ground state structure and excitation energies, oscillator strengths and transitions vectors can be determined from the linear response [
19]. DFT and TD DFT methods have been usually shown to provide reasonable and consistent with experimental data results with relatively low computational costs. Previously performed theoretical studies on phenolic compounds with the hybrid Becke’s three parameters with Lee–Yang-Parr (B3LYP) functional [
20,
21,
22] give results consistent with experimental data [
23,
24,
25]. The choice of the applied exchange-correlation functional and the utilized basis set is usually the compromise between the time of computations and the quality of the obtained results. Although previously published studies [
18] showed that application of 6-31G (d,p) basis set usually gives reasonable molecular geometries of chalcone molecules, the incorporation of diffuse and polarization functions is mandatory for UV spectral studies, especially for chromophores with extended π electrons [
26].
It is well known that the type of solvent may affect UV–Vis spectra of chromophores, therefore, the incorporation of solvent effects is very important in the theoretical prediction of the absorption spectra. Polarizable Continuum Model (PCM) is the most frequently incorporated solvation model in theoretical studies of flavanones and chalcones structurally similar to the compounds under study [
14,
15]. The PCM model [
27,
28] is based on the assumption that solute is embedded in a shape-adapted cavity within solvent modelled as a dielectric continuum of defined dielectric constant.
To the best of our knowledge only few previously published studies have been devoted to the structure and properties of the studied hydroxyflavanones and their corresponding chalcones. Wróblewski and et. [
15] have applied both experimental (absorption and fluorescence spectroscopy) and theoretical (TD DFT(PCM)) methods to describe the photophysical characteristic of 7-HF in methanol, ethanol and acetonitrile.
The structure and spectral characteristic of 2′hydroxychalcone and its derivatives with different alkyloxy groups at position 4′ were previously extensively investigated by Serdiuk et al. [
16]. The authors concluded that generally both in crystals and solution there is a little impact of substituents on absorption spectra of chalcones. The authors revealed that 2′hydroxychalcones form crystal lattices with different packing patterns. Hydroxychalcones are planar in gas and crystalline phase and they do not change the conformation upon excitation. The authors proposed that in liquid media several processes for the excited state deactivation such as isomerization in the S
1 state (geometrical changes), intersystem crossing and conical intersection are possible. The authors concluded that molecular conformation is the key factor determining the fluorescent properties of the phototautomer keto species formed by the excited state intramolecular proton transfer (ESIPT). The ESIPT process in 2′ hydroxychalcones, which is facilitated by the presence of hydrogen bond between hydroxy and carbonyl groups, was also studied previously [
29].
The aims of this work were to investigate influence of pH on acid–base and spectral profiles of flavanone (F), 2′-hydroxyflavanone (2′HF), 6-hydroxyflavanone (6HF) and 7-hydroxyflavanone (7HF) and to reveal their interactions with CT DNA. Potentiometric titration, UV–Vis spectroscopy were used to characterize physicochemical properties of these flavanones and to propose the mode of interaction with CT DNA. Cyclic voltammetry was applied to evaluate antioxidant potentiality of studied flavanones. Additionally, a theoretical DFT (B3LYP) method has been used to disclose electronic structure and properties of the compounds. In particular molecular geometries, proton affinities and pKa values have been determined. Moreover, the time dependent DFT (TD DFT(B3LYP)) calculations have been performed on the optimized geometries to provide insight into the energies and nature of the electronic transitions which contribute to the absorption spectra of F, 2′HF, 6HF and 7HF.
4. Conclusions
The results of our research have shed some light on clarification of different biological activity of the flavanones studied. As the spectral results both experimental and theoretical indicate the compounds have revealed different susceptibility to microenvironment, i.e., to components of the solution or changes in the concentration of hydrogen ions whose presence is of crucial importance in cellular compartments. This is especially seen in spectral profiles of 6HF (
Figure S2). This finding correlates with its stronger cytotoxic effect compared to the other studied flavanones. It leads to the conclusion that this compound can react with cell components, such as enzymes or apoptosis proteins.
Based on the calculated HOMO and LUMO energies, together with cyclic voltammetry data, it was possible to make predictions of reactivity of 6HF, 2′HF 7HF and F. Analyzing the calculated quantum chemical descriptors, it can be suggested that based on the lowest HOMO-LUMO gap and the lowest IP, 6-hydroxyflavanone is expected to be the best antioxidant in this set of monohydroxy-flavanones. Structure–activity relationships observed for antioxidant activity and DNA interactions suggest that 6-hydroxyflavanone can protect DNA against oxidative damage more effectively than flavanone, 2′-hydroxyflavanone or 7-hydroxyflavanone.
Weak interactions with CT DNA indicate that the anti-tumor activity of these flavanones is unlikely to occur by destroying the structure of DNA, but rather through their interaction, e.g., with apoptotic proteins in cancer cells.
To summarize, we have shown that even such subtle changes as altering the location of the OH group from the C6 position to the C7 position in 6HF and 7HF, respectively, lead to significant changes in the spectral profile, which means changes in the electronic structure of the flavanone rings, and this is reflected in their biological activity.