1. Introduction
Flavonoids are the most common group of polyphenolic compounds and are found in a huge variety of plants [
1]. They feature low molecular weight and exert effects such as modulating reactive oxygen species (ROS) levels, influencing the plant hormone auxin′s transport, and act on defense mechanisms and allelopathy [
2]. They are divided into the following classes according to substitutions: flavones, flavonols, chalcones, aurones, flavanones, isoflavones, catechins and anthocyanidins. Flavones and flavonols are biosynthetically produced. It can be said that the flavonols are flavones with a hydroxyl substitute in position C3. Their analysis, synthesis, and reactions share therefore a common theoretical basis [
3].
Several studies have demonstrated that flavones have anticancer activity [
4,
5,
6,
7], antioxidant [
8,
9,
10], antitumor [
11,
12,
13], anti-inflammatory [
9,
14,
15,
16], cardioprotective [
8,
17,
18]; antiviral [
19,
20,
21], neuroprotective [
7,
9], antibacterial [
22,
23,
24], and induction of apoptosis in leukemic malignant cells [
25]. These biological activities depend on the chemical structure and the various substituents that these molecules have; the basic backbone can undergo a number of modifications, such as glycosylation, esterification, amidation, and hydroxylation, among others that modulate the polarity, toxicity and intracellular actions of these compounds [
26].
Software for in silico screening is intended to perform virtual study of molecules, and includes tools that predict theoretical selectivity and recognition for a given site of action or bioreaction, and are generally associated with large databases of small molecules for structural comparison [
27,
28,
29]. Computational prediction models, also called predictive tools, play a prominent role in the repertoire of methodologies guiding pharmaceutical technological research. These devices are used to study existing and hypothetical substances, predicting results of pharmacological, pharmacokinetic and toxicological behavior [
30,
31]. Such predictive tools have already been recommended by regulatory agencies for studies of technological development in order to verify the theoretical toxicity of substances in the mammalian metabolic environment [
32], in addition to allowing the creation of a database of relations between chemical structure and biological activity (SAR) [
33].
The objective of this work was to perform in silico screening of theoretical pharmacological, pharmacokinetic, and toxicological activities as well as to investigate the antibacterial, oxidant and antioxidant effects of flavone, 3-hydroxyflavone, 5-hydroxyflavone and 6-hydroxyflavone (
Figure 1); evidencing the relationship between chemical structure and observed pharmacological effect, and consolidating the use of computational chemistry to characterize substances with therapeutic potential.
3. Discussion
PASS revealed various biological possibilities: probable agonist action for cell membrane integrity and inhibition against membrane permeability; probable inhibition of kinases, antimutagenic activity and metabolic influence on cytochrome P450 enzymes, both as substrate and as inducer; in addition: flavone, 5-hydroxyflavone and 6-hydroxyflavone were assigned inhibition of peroxidase and oxidoreductase, and vasoprotective potential.
The flavonoids presented possible antitumor activity; flavone, 5-hydroxyflavone and 6-hydroxyflavone had predicted antitumor potential via inhibition of the Pin1 gene, and all three hydroxylated derivatives showed theoretical TP53 gene increases.
There are many mechanisms proposed for the effects promoted by flavonoids during the initiation and promotion stages of carcinogenicity; the principal effects are given as follows: down regulation of mutant p53 protein, cell cycle arrest, tyrosine kinase inhibition, inhibition of heat shock proteins, estrogen receptor binding capacity and inhibition of Ras proteins expression [
34].
The Pin1 gene (Peptidyl-prolyl
cis–
trans isomerase NIMA-interacting 1) has demonstrated certain functional polymorphisms associated with cancer risk; the gene regulates conformation of phosphorylation sites, and it has been involved in multiple oncogenic signaling pathways as a critical catalyst. TP53 is a tumor suppressor gene known as “the cellular gatekeeper of growth and division”, it acts controlling cellular growth and by inducing important genes in cycle arrest and apoptosis following DNA damage [
35,
36].
When protein kinases (PKs) are deregulated pathological conditions may originate defects in phosphorylation leading to uncontrolled cell division, inhibition of apoptosis, and other abnormalities. Knowing this, the inhibition of these enzymes is as a promising strategy against cancer, and various studies have shown that flavonoids are capable of inhibiting a number of protein kinases from differing cellular signal pathways, such as: tyrosine kinase (PTK), serine/threonine kinases, and phosphatidylinositol 3-kinase (PI3K) [
37].
One of the targets that flavones can interact with is phosphatidylinositol 3-kinase. The PI3K pathway is one of the most frequently activated signaling routes in human cancers, occurring in 30–50% of the cancerous cells [
38]. The PI3K is an enzyme generally regulated by growth factors; when activated, PI3K adds a phosphate group to phosphatidylinositol 4,5-bisphosphate (PIP2), generating phosphatidylinositol 3,4,5-triphosphate (PIP3), an intra-cellular messenger. Akt, also known as protein kinase B (PKB), interacts with PIP3 and translocates to the plasma membrane, Akt is activated downstream of PI3K and has multiple targets that are extremely important in the balance control between survival and apoptosis. Some studies indicate in cancer patients, that activation of the PI3K/Akt pathway occurs because its components are targeted for amplification, mutation and translocation more frequently than any other pathway [
39,
40].
Lee et al. [
41] demonstrated that apigenin (4′,5,7-trihydroxyflavone), a natural flavonoid, inhibits HGF-induced (Hepatocyte growth factor) which controls invasive growth of MDA-MB-231 human breast cancer cells, including their motility, scattering, migration, and invasion; through blocking the PI3K/Akt pathway. Woo et al. [
42] also testing natural flavonoids investigated the effect of chrysin (5,7-dihydroxyflavone) on the apoptotic pathway in U937 human promonocytic cells; it was shown that chrysin induces apoptosis in association with the activation of caspase 3, and the Akt signal pathway plays a decisive role in chrysin-induced apoptosis in U937 cells. In the same study, they showed apoptosis increases when Akt phosphorylation in U937 cells was inhibited by the specific PI3K inhibitor; significantly LY294002, enhanced apoptosis.
The flavones tested in this work present certain structural similarities with the compounds mentioned above, and have also demonstrated a theoretical inhibitory effect on protein kinases; able to interact with PI3K/Act pathway and ERK (extracellular signal regulated kinase).
Flavone was found to have an antimicrobial effect, with an MIC of 100 μg/mL, against the strains
E. coli 2536,
E. coli 101, 103
E. coli,
E. coli 105,
P. aeruginosa ATCC 23243, and
S. aureus ATCC 25619, Gram positive and Gram negative bacteria with an MIC of 200 μg/mL for the hydroxylated flavone derivatives, especially 3-hydroxyflavone that inhibited the growth of all bacterial strains tested (
Table 5 and
Table 6). This noted wider effect can be attributed to the presence of a hydroxyl in position 3 of the benzopyran carbon ring of the flavonoid, next to the carbonyl. It should also be noted that flavone and 5-hydroxyflavone at a concentration of 25 μg/mL showed antibacterial effect on strains of
E. coli (103, 105, and 108). This concentration was well below that used for the standard drug chloramphenicol (100 μg/mL).
With regard to the antibacterial effect, flavone presented a bactericidal concentration of 200 μg/mL against strains of P. aeruginosa ATCC 8027, S. aureus ATCC 25619 and E. coli 104, while the other flavonoids were bacteriostatic at 200 μg/mL.
Ríos & Recio [
43] posit avoiding evaluation tests for antibiotic activity with extract concentrations of greater than 1000 μg/mL, or 100 μg/mL for isolated substances. Antimicrobial potential is interesting when detected at concentrations below 100 μg/mL for extracts, or 10 μg/mL for active ingredients. To assign positive activity for excessively high concentrations is to be avoided. Following this evaluation criterion, the antibiotic activity of flavonoids is to be understood as moderate.
In QSAR evaluation study with flavonoids it was found that good antibacterial activity structure requirements include hydroxyl groups in positions C-3, C-5, C-7 and C-3′, the C-2, 3 unsaturated double bond and a carbonyl group at C-4 is essential, while the presence of the hydroxyl group on C-6, can lower the antibacterial activity [
44]. The hydroxyl group normally works as a lead donor H, the presence of an electron donor group in conjunction with the group α, β-unsaturated, can promote electronic interactions, which decrease your bactericidal effect, observing due to the presence of variations in the structure of the compound and the replace mode [
44,
45]. Still, lipophilic flavonoids can also disrupt microbial membranes [
34], so the high lipophilicity of flavone results in increased hydrophobic interaction by promoting a better antibacterial effect.
Lovewell et al. [
46] identified for the first time that the PI3K/Akt pathway mediates the motility-driven phagocytosis of
P. aeruginosa, and the gradual loss of bacterial flagellar motility is proportional to the degree of Akt activation in host cells, in parallel to the phagocytic susceptibility. This means that therapies using PI3K/Akt pathway inhibitors could act as bactericides, preventing phagocytosis and motility.
Since the compounds present in this work had a theoretical effect on kinases, they could act in such a way on
P. aeruginosa. Flavonoids are able to modulate the activity of enzymes and affect the behavior of many cellular systems, exercising beneficial effects on the body [
47]. Several studies have identified and attributed to flavonoids antioxidant potential. Ghasemzadeh et al. [
48]. Reported that the total phenolic compounds and flavonoids in
Halia bara possess potent antioxidant activities.
Zhao [
49] suggests that the action of propolis comes from its flavonoids′ ability to interact, having lipophilic characteristics, with the bacterial plasma membrane, causing disorder. Perhaps it is through this mechanism that flavone acts in promoting bactericidal activity, since it is among the tested, the flavonoid featuring a greater cLogP (3.74), giving greater lipid solubility and allowing the same to cross the membrane. However, other mechanisms might justify the antibacterial action observed, such as inhibition of DNA gyrase, an enzyme responsible for separating the double helix during DNA replication [
50,
51].
To combat oxidative stress, cells and tissues have enzymatic and non-enzymatic antioxidant defense systems. However, under conditions of extreme stress such endogenous systems are not sufficient; making the support provided by antioxidants of exogenous origin necessary [
31]. In this context, the red blood cells enjoy wide use in studies of oxidative stress, being a simple representative cell model. Their membranes possess the functions of other cells, such as active and passive transport and electrochemical gradients. Hydrogen peroxide (H
2O
2), induces changes in the form of these cells, characterized by bubbles or bulges on the cell membrane, which indicate oxidative damage [
52].
From analysis of the results expressed in
Figure 2a–d it was possible to assign non-concentration dependent antioxidant effect to flavone, 3-hydroxyflavone, 5-hydroxyflavone and 6-hydroxyflavone in all concentrations evaluated using reductions in hemolysis as induced by hydrogen peroxide (H
2O
2), this as compared to the control group (Hb + H
2O
2), in some situations these flavonoids showed more antioxidant potential than vitamin C.
Another protocol used to investigate the antioxidant potential of these flavonoids was the erythrocyte oxidative agent phenylhydrazine, revealing the percentage of methemoglobin formation in relation to hemoglobin (% metHb/Hb).
The
Figure 3a,
Figure 4a,
Figure 5a and
Figure 6a show respectively that flavone, 3-hydroxyflavone, 5-hydroxyflavone and 6-hydroxyflavone did not oxidize red blood cells when compared to the negative control group (Hb), and in turn presented antioxidant activity in the presence of phenylhydrazine, seeing the reduced percentages of methemoglobin formation in relation to hemoglobin (% metHb/Hb) (
Figure 3b,
Figure 4b,
Figure 5b and
Figure 6b) as compared to the positive control group (Hb + Ph).
The search for new antioxidant agents is important because the oxidative stress causes peroxidation of membrane lipids, aggression to proteins in tissues and membranes, enzymes, carbohydrates and DNA. These damages have been related to the pathogenicity of some diseases, such as atherosclerosis, neuronal degeneration, cancer, rheumatoid arthritis, diabetes mellitus, inflammation and vascular disease [
53,
54,
55].