Among natural products, flavonoids are widely distributed in plants, showing a broad range of biological activities [1
], like antioxidant [2
], anticarcinogenic [6
], and antimicrobial [8
] effects. The basic flavonoid structure is represented by a C6-C3-C6 carbonated skeleton with different bonds between the phenylpropanoid and acetate units, being classified as isoflavonoids, neoflavonoids, and flavonoids [9
]. They can also be hydroxylated, glycosylated, sulfated, and methylated, resulting in a great variety of different compounds [9
]. Based on this structure diversity, and their activity against Gram-positive and Gram-negative bacteria [9
], this family of compounds constitutes an especially interesting group for the development of new antibacterial drugs.
An antibacterial compound requires a special set of physicochemical properties to access the site of action inside the bacterial cell [15
]. The cell wall represents a complex barrier to the entrance of compounds. Gram-positive bacteria possess a thick peptidoglycan layer and, in the case of Gram-negative bacteria, in addition to the inner membrane and the peptidoglycan layer, an outer membrane represents an additional barrier for many small molecules. A compound whose site of action is inside the cell must interact with the cell membrane to gain access to its target in the required critical concentration, and a lipophilic moiety is important for this interaction [16
]. The permeability of small molecules is correlated with their solubility in nonpolar solvents and their solubility in water [18
]. To cross the bacterial membrane, the small molecule is first solvated in the extracellular media, immersed in the alkylated part of the lipid bilayer, finally diffusing to the cytoplasmic side, where it is again solvated. This behavior can be related experimentally with the partition coefficient of the compound in a non-polar/aqueous phase, and theoretically with the lipophilicity. Using this latter approach in filifolinol derivatives, good qualitative correlation was shown between lipophilicity and antimicrobial activity [19
], the relationship between the lipophilicity of flavonoid phytoalexins analogues and the growth inhibition of two fungi (Aphanomyces euteches
and Fusarium solani
), and with the minimal inhibitory concentration against the Gram-positive bacteria Streptococcus faecium
]. In the last cases, they found that the flavonoid activity was different depending on the presence of one hydroxyl group, and that at least some lipophilicity was needed for both the antifungal and antibacterial activities. Retrochalcones were more active against bacteria when the hydrophobicity was improved by the addition of prenyl groups on ring B [20
]. Structurally, the position and number of hydroxyl groups on the flavonoid rings were also important for the antioxidant activity [2
In the design of novel drugs, a good understanding of the properties that are responsible for their activity is an important goal in their development. A second important aspect for the potential antibacterial activity of flavonoids is their permeation through the cell walls of microorganisms [15
] and their interaction with the cell membrane [22
]. This property is associated with their solubility in the lipid bilayers that protect bacterial cells, and may be evaluated by the assessment of the drug’s lipophilicity [17
] by means of a fluorescence polarization method, analyzing the interaction of flavonoids with liposomal membranes, showing that the antibacterial activity of flavonoids against Escherichia coli
depends on the molecular hydrophobicity and the charges on C-3 atom.
The requirement of a hydrophobic behavior of these molecules is in contrast with their need of having hydrophilic phenolic groups for their activity. A proper method of evaluating these compounds should incorporate these opposing features in the measurement of some physico-chemical properties that might correlate with their activity. The determination of their diffusion coefficients in a protic solvent takes into account these opposing factors. Their diffusion in a hydroxylic solvent should depend not only on their size, but also on their intermolecular interactions with the solvent. The presence of hydrogen-bond-forming groups in the molecule increases these interactions, whereas hydrophobic groups tend to reduce them.
In previous publications the diffusion coefficients of some flavonoids in methanol were measured by the Taylor-Aris technique [23
], which has been used for estimating drug partition in biological systems. The results allowed an analysis of the solute-solvent interactions of these systems to be made, that was supported by dynamics simulations [24
]. There is no rigorous data published elsewhere about a correlation between the lipophilicity and antibacterial activity of flavonoids, and how the lipophilicity affects compound-membrane interaction.
In the present communication, we applied the Taylor-Aris technique to measure the diffusion coefficients and the theoretical lipophilicity estimation of flavonoids, specifically flavones and flavanones, searching for correlations between these physico-chemical parameters and their activity as antimicrobial agents against Gram-negative and Gram-positive bacteria.
The selection of antibacterial activity determination methods is very important when the compounds to be tested are insoluble in water, as is the case for the flavonoids. In solid media, the paper disk and the cylinder methods require the diffusion of the compounds into the aqueous agar media [29
]. To avoid this problem we used the direct assay method, depositing aliquots of the compound over the bacterial lawn, also allowing the rapid evaporation of methanol, the solvent used in this case. On the other hand, the antibacterial activity determination in liquid media has the problem of observed opalescence due to the insolubility of the compounds in aqueous solutions that interfere with the optical density when bacterial growth is quantified. This was solved by subtracting each optical density (O.D.) obtained with the different compound concentrations used in the assay in culture media. Despite this, at higher concentrations, it is possible that aggregations are formed between the compounds and bacteria, or forming a cumulus of dead and live bacteria as reported using galangin with S. aureus
The antibacterial activity of the compounds galangin, naringenin, and quercetin are consistent with MIC values published using different S. aureus
]. It is noteworthy that this is the first report of antibacterial activities against S. aureus
-methylisorhamnetin, and pinocembrin. In the case of 3,7-O
-dimethylgalangin, antibacterial activity had only been informed with MIC values of 0.050 μg/μL and 0.1 μg/μL against methicillin-susceptible S. aureus
(MSSA) and methicillin-resistant S. aureus
(MRSA), respectively [31
Results in solid and liquid media using the microdilution method showed that Gram-positive bacteria were slightly more susceptible to the active flavonoids than Gram-negative bacteria, as determined by the MIC. This may be due to differences in the cell wall structure between both types of bacteria [15
]. The interaction of antibacterial compounds with the cytoplasmic membrane defines the success or failure in performing their inhibitory activity. For this reason, their physico-chemical properties, and specifically their lipophilicity, are crucial. Non-hydrophobic compounds would not cross the membrane, except through special pores, and highly-hydrophobic compounds would join too strongly to the phospholipid bilayer. Our results show that there is a close relation between lipophilicity and antibacterial activity, determined by the diffusion coefficient supporting a prediction analysis of active compounds. The most active compounds against the Gram-positive bacteria B. subtilis
-methylgalangin, galangin, pinocembrin, naringenin, and 7-O
-methyleriodictyol, showing that the diffusion coefficient should be in the range of 9.4 × 10−10
to 12.3 × 10−10
/s, which was further supported by the lipophilicity value range. B. subtilis
showed a wider range compared to E. coli
. In the latter case the active compounds were only two, 3-O
-methylgalangin and galangin. These results may be explained by the differences in the external structure of both types of bacteria, as mentioned in the introduction.
To simplify the analysis of the chemical structure and antibacterial activity, from the relationship based on their structural classification, the compounds were grouped into flavanones and flavones. Flavanones, which differ from flavones by the lack of the hetero-ring double bond, are not planar compounds. Flavones are almost planar molecules but, in contrast, flavanones are not planar, with the exocyclic phenyl ring almost perpendicular to the rest of the molecule. Despite the few compounds tested, the comparison between a flavone and flavanone with similar lipophilicity and oxygenated substitution patterns in the A and B rings (e.g., pinocembrin and 3-O-metilgalangin) showed that flavones have higher antibacterial activity. In addition to the planar structure, as concluded from the marked antibacterial activity of flavone, itself, several structural requirements emerged from this study.
The flavanones naringenin, 7-O-methyleriodictyol, and pinocembrin showed lower activity against Gram-positive bacteria compared with flavones. In Gram-positive bacteria the flavanones were more active, but when the number of hydroxyl groups in the compounds is greater, e.g., in narigenin and 7-O-methyleriodictyol, which have three hydroxyl groups, the compound showed less activity, while pinocembrin, with two hydroxyl groups, showed the lowest MIC. This trend was also evident with the Gram-negative bacteria. The above shows that the number of hydroxyl groups should not be an important factor, but their position proves to be a relevant factor, showing that the presence of two well-defined zones in the structure, a hydrophobic and a hydrophilic one, are necessary. Examples of this are 7-O-methyleriodictyol and pinocembrin, which are active against both types of bacteria and, moreover, pinocembrin is the most active, and it has hydroxyl groups in C-5 and C-7 on ring A, leaving ring B free of substituents. This suggests that the presence of two hydroxyl groups on ring A and none on ring B is a major contributing factor towards antibacterial activity. Pinocembrin also presents the highest distribution coefficient (D) and lipophilicity (log P) among the flavanones that were studied.
Among the flavones that were analyzed, the importance of the presence of hydroxyl groups for the antibacterial activity was clearly shown by comparing the activity of the series of galangin with one, two, and three hydroxyl groups in the structure, which is consistent with the structure-activity analysis recently published [33
]. The most lipophilic compound, and less active as an antibacterial agent, has only one hydroxyl group (3,7-O
-dimethylgalangin), whereas galangin was the most active, with three hydroxyl groups in its structure, followed by 3-O
-methylgalangin, which has an intermediate lipophilicity in this series. Analyzing the remaining flavones, quercetin showed low activity, with five hydroxyl groups and high lipophilicity. As indicated before, for the compound to reach its site of action inside the bacterial cell a high hydrophilic nature would prevent it from crossing the cytoplasmic membrane. Thus, there is no lineal relation between lipophilicity and activity; instead, there is a range of lipophilicity in which the compounds can be more active, or there may be other structural factors, which could also be involved in the antibacterial activity.
The above results are consistent with the trend for flavanones where, besides the number of hydroxyl groups, their position plays an important role in the activity. This becomes apparent with the requirement of high amphipathicity of the molecule, with well-spaced hydrophobic and hydrophilic regions, which seem to be essential for the activity of the compounds. A clear example is that of galangin and its methoxylated derivative (3-O
-methylgalangin), where it was found that the polar zone with the hydrophilic characteristics is located on ring A, while the aromatic ring B lacks substituents, constituting a lipophilic region of the molecule. It is known that the mode of antibacterial action of galangin in S. aureus
is through the disruption of the integrity of the cytoplasmic membrane producing loss of potassium [9
]. A further study showed that galangin causes aggregation of the bacterial cells of S. aureus
, involving the cytoplasmic membrane as the target site for the activity of this compound [30
The presence of a hydroxyl group and a methoxy group on ring B (3-O
-methylisorhamnetin) causes a drastic decrease in activity compared to galangin; the same is true for the compound with two hydroxyls on ring B (quercetin) emphasizing the importance of these groups. For flavonoid activity in eukaryotic cells, has stressed that the interaction with the cell membrane is important [22
]. Such interaction with lipids is, in most cases, limited by the polar region of the phospholipid bilayer, but the penetration depth of these compounds into the membrane depends on its structure. Most flavonoids decrease membrane fluidity. The main factor governing flavonoid-phospholipid interactions seems to be the lipophilicity of these molecules. It also appears that the presence of two hydroxyl groups located strategically on ring A and the absence of polar groups on ring B are of great importance for the antibacterial activity of flavonoids, because these groups bind strongly to the polar phospholipids region in the bacterial membrane, leaving the hydrophobic region with B ring facing the inside of the bilayer, interacting with the alkyl chains of the phospholipids. Such structural factors would explain the disruption of the membrane resulting in the bacteriolytic action of these compounds.