Antimicrobial Activity and Chemical Composition of Essential Oils from Verbenaceae Species Growing in South America

The Verbenaceae family includes 2600 species grouped into 100 genera with a pantropical distribution. Many of them are important elements of the floras of warm-temperature and tropical regions of America. This family is known in folk medicine, and its species are used as digestive, carminative, antipyretic, antitussive, antiseptic, and healing agents. This review aims to collect information about the essential oils from the most reported species of the Verbenaceae family growing in South America, focusing on their chemical composition, antimicrobial activity, and synergism with commercial antimicrobials. The information gathered comprises the last twenty years of research within the South American region and is summarized taking into consideration the most representative species in terms of their essential oils. These species belong to Aloysia, Lantana, Lippia, Phyla, and Stachytarpheta genera, and the main essential oils they contain are monoterpenes and sesquiterpenes, such as β-caryophyllene, thymol, citral, 1,8-cineole, carvone, and limonene. These compounds have been found to possess antimicrobial activities. The synergism of these essential oils with antibiotics is being studied by several research groups. It constitutes a resource of interest for the potential use of combinations of essential oils and antibiotics in infection treatments.


Introduction
The Verbenaceae family includes 2600 species grouped into 100 genera with pantropical distribution. The most significant number of species is found in Latin America where they occur in a wide array of ecosystems. This family involves herbs, shrubs, and a few trees. They are an important element in the flora of South America [1].

Essential Oils from the Verbenaceae Family
Essential oils are heterogeneous mixtures that may contain many compounds at different concentrations. Each EO is characterized by some major compounds, which can reach high levels compared to other compounds present in trace amounts.
It is known that the occurrence of secondary metabolites with similar biological activities can be expected in phylogenetically related plants, which may contribute to the implementation of more rational approaches for the search of new substances with potential economic interest [54]. This section provides information on the chemical composition of five genera and several species of different genres of the Verbenaceae family.
The Lantana species presented β-caryophyllene in high concentration, with cubebene, elixene, and phellandrene as minor compounds [59]. The β-caryophyllene is a chemical marker for species belonging to the Lantana genus [60]. Lantana camara is the most widespread species of this genus. The chemical composition of L. camara EO plays a role in its biological activity; the β-caryophyllene and (E)-nerolidol chemotypes showed antimicrobial and cytotoxic activities [61].
The compositions of the EO of the aerial parts of some species of the Aloysia genus presented oxygenated sesquiterpenes as the main components (between 40% and 50%) followed by sesquiterpenic molecules (30-34%) [70]. The EO from the leaves of A. gratissima species, examined in Brazil, Uruguay, and Argentina, contained limonene, sabinene, α-pinene, β-bisabolene, and copaenol; the EO from the flowers presented a high percentage of pulegone (65.8%) and other featured components, such as limonene, spathulenol, α and β-thujene, dihydrocarvone, and menthone [71].
There are very few data related to the chemical composition of the EOs of species belonging to the genus Stachytarpheta. The EO of Stachytarpheta gesnerioides is mainly composed of guaiol (53.5%), α-pinene (16.1%), and isocaryophyllene (1.7%) [76]. In the composition of the EO from the leaves of Stachytarpheta mutabilis, oxyquinone sesquiterpenes, such as those of the eudesman type, are present in a greater percentage [77].

Antimicrobial Activity
Aromatic plants have great importance for the pharmaceutical industry, although synthetic products replaced many of them. Plant species show inhibition of bacteria, fungi, and yeasts. Nowadays, the emerging problem of antibiotic resistance is a driving force behind multiple studies on the potential antibacterial activity of EOs and the development of novel preventive or therapeutic strategies for health.
Many aromatic plants are practically immune to the attack of herbivores because of the presence of bioactive metabolites. This fact makes them attractive for study in search of new antimicrobials. The active compounds may be present in the stems, leaves, roots, flowers, or fruits. Regarding the study of the Verbenaceae family, most of the works involved the leaves as a source of EO, but some studies also used flowers.
The diversity of techniques used makes it difficult to compare results from different groups of researchers. Several are the factors that influence the data found in published works related to this topic, namely, microbiological methods, techniques of EO extraction, plant part involved in the extraction, harvesting season, climatic and environmental conditions where the plants were cultivated, and others. Consequently, the differences observed between articles could not possibly reflect a real difference between EOs' features. It has been found that there is no consensus about both the methodology for the evaluation of the antimicrobial activity of a natural product, and an acceptable value for the minimal inhibitory concentration (MIC). Deep discrepancies among researchers have been found when considering antibiotic-like concentrations or good antimicrobial potential, even when the EO activity consists in a weak inhibition. We consider that a comparison between the MIC values of EOs and commercial antibiotics is not applicable, because the former contain a mixture of compounds which can interact among them, whereas the latter are pure compounds. Aligiannis et al. [78], for example, classified the antibacterial activity of plant extracts on the basis of MIC results, indicating as strong inhibition that corresponding to MIC up to 500 µg/mL, moderate inhibition that characterized by MIC between 600 µg/mL and 1500 µg/mL, and weak inhibition that observed with MIC above 1600 µg/mL. In contrast, Holetz et al. [79] argued that MIC values lower than 0.1 mg/mL represent strong antimicrobial action, values between 0.1 mg/mL and 0.5 mg/mL indicate moderate antimicrobial activity, values between 0.5 and 1.0 mg/mL indicate weak action, while values above 1.0 mg/mL indicate inactive products. The analysis of the MIC values found in the studied EOs allows us to suggest that values less than or equal to 0.5 mg/mL represent strong activity, while those between 0.5 mg/mL and 1.0 mg/mL indicate moderate action. The values between 1.0 mg/mL and 2.0 mg/mL indicate weak action and those greater than 2.0 mg/mL should be considered as corresponding to lack of activity. This appreciation can be useful, since the previous work focused mainly on plant extracts and not on essential oils. Table 2 presents studies on the antimicrobial activity of EOs from the Verbenaceae family against human pathogens, carried out in South America in the last two decades. It should be noted that the broth microdilution test is the most used method to determine the MIC. These data are detailed in the following paragraphs.
According to Sartoratto et al. [80], the EO from A. triphylla showed inhibition values between 0.05 and >2 mg/mL against Gram-positive bacteria but it was not active against Gram-negative bacteria. However, Duarte et al. [81] reported that A. triphylla inhibited 12 Escherichia coli serotypes, with MIC values between 400-1000 µg/mL, showing moderate activity. A. polystachya showed inhibition values between 3.64 and 29.13 µg/mL against Gram-positive and Gram-negative bacteria, but it was not active against Pseudomonas aeruginosa [82]. A. gratissima showed inhibition values between 1000 and 4000 µg/mL against Gram-positive and 2000-4000 µg/mL against Gram-negative bacteria. The minimal bactericidal concentration (MBC) was twice the MIC in most of the cases and was the same as the MIC only for E. coli [8]. Aloysia sellowii showed inhibition values between 1.7 and 16 mg/mL against Gram-positive bacteria and between 6.7 and >20 mg/mL for Gram-negative bacteria. The MBC values were between two and three times the MIC. Aloysia sellowii was active against yeasts with MIC values between 4-16 mg/mL and Minimal Letal Concentration between 4->20 mg/mL [14].
Other species mentioned in Table 2 were studied by means of the disk diffusion assay. Disks of 6 mm of diameter were impregnated with 10 or 15 µL of EO, resulting in inhibition halos between 8 and 20 mm diameter. Aguiar et al. [89] considered that halos with a 10 mm diameter represent a good inhibition. According to the observed results in the literature, halos with diameters larger than 20 mm are rare. However, there are reports with these values for L. origanoides [90,91], Lippia gracilis [92] and Lippia grandis [43]. The target bacteria generally used in this type of studies were S. aureus, S. epidermidis, Bacillus cereus, and E. faecalis (Gram-positives), E. coli, P. aeruginosa, K. pneumoniae, and Salmonella (Gram-negatives). The use of ATCC strains facilitates the comparison of the antibacterial potency between plants of the same species that grow in different regions. However, the use of clinical isolates gives a valuable contribution in this field, since it gives a more realistic scenario of the activity of EOs against pathogen strains.

Antimicrobial Synergism
Over the last decades, interactions between natural products and commercial antibiotics have been comprehensively studied. Many researchers have demonstrated the ability of natural products to regulate antibiotic activity possibly exerting a synergistic effect.
The checkerboard test evaluates the effect of interactions between two antimicrobial substances. This assay is one of the most commonly used to determine synergism. The MIC values of combinations are registered at one time point [106], and the fractional inhibitory concentration (FIC) index for the two antimicrobial substances are calculated. Sometimes, the results of this assay are interpreted by plotting an isobologram. Another test used to determine synergism is the time-kill assay. It involves measuring the number of viable bacteria in a liquid medium in the presence of a particular combination of antimicrobial substances at different time points. Although time-kill curves are not widely used to study antibacterial interactions, they can be considered a clinically relevant model if the concentrations used represent those achieved at the site of an infection [107]. As previously mentioned, for the determination of antibacterial activity, methods used to evaluate interactions between EOs and antibiotics differ widely, and this makes data comparison difficult. However, the use of FIC indexes allows comparisons of the results. The development of a more standardized method of serial passaging in sublethal concentrations of EO would enable a better investigation of the possible loss of sensitivity or cross-resistance [108].
The commercial antibiotics and bacterial strains most frequently used to evaluate a synergistic action are shown in Table 3. The most relevant results are described below.
Many examples of synergism can be found in the literature regarding Lantana species. The concentration of neomycin decreased 50% against multiresistant E. coli when this antimicrobial was combined with L. caatingensis EO. Similarly, there was a 75% decrease in the concentration of amikacin against S. aureus ATCC 12692 when this EO was present [16]. L. camara EO at 50 µg/mL increased the activity of amikacin up to 65% against P. aeruginosa and up to 29% against S. aureus. When this EO was combined with gentamicin, the antibiotic efficacy increased to 21% against P. aeruginosa and at none effect was detect against S. aureus [24]. Lantana montevidensis EO at 50 µg/mL improved the activity of gentamicin by 12% against P. aeruginosa ATCC 15442 and by 10% against S. aureus ATCC 12692. In addition, when this EO was combined with amikacin, the antibiotic efficacy increased up to 102% against P. aeruginosa ATCC 15442 and to 29% against S. aureus ATCC 12692 [25]. The combination of neomycin with L. montevidensis and L. camara EOs showed no interaction of the two components against S. aureus ATCC 6538 [26].
Likewise, Lippia species exerted several synergistic effects. The presence of 12% L. alba EO increased between 12.5% and 35.7% the activity of erythromycin against two S. aureus ATCC [37]. Lippia gracilis showed a modulatory effect on aminoglycoside activity. A reduction of the MIC value of gentamicin and amikacin against two E. coli strains and S. aureus were observed [40]. Moreover, the addition of 128 µg/mL of L. origanoides EO to the growth medium did cause a 10-fold decrease in the MIC of neomycin (2500-248 µg/mL) and amikacin (788-78 µg/mL). This demonstrated a synergistic effect between L. origanoides EO and aminoglycosides against the methicillin resistant Staphylococcus aureus (MRSA) strain [46].  [87] The presence of L. sidoides EO at 50%, 25%, 12% and 6%, produced an increase of 429.41%, 349%, 256.82% and 21.53% in gentamicin activity against a S. aureus strain. The activity of amikacin and neomycin was enhanced when either antibiotic were combined with this EO [52]. Veras et al. [87] combined L. sidoides EO with aminoglycosides (gentamicin and neomycin) and β-lactams (penicillin G and ceftriaxone) and demonstrated indifferent and synergistic effects depending on the bacteria tested. The combination of the EO with aminoglycosides was synergistic on S. aureus and P. aeruginosa (the MIC was reduced four times). Synergism was also detected when EO and gentamicin were used against K. pneumoniae (the MIC decreased from 32 to 1 µg/mL). This mix showed no effects on the other bacteria. Regarding the interactions between this EO and β-lactams, synergism was detected against S. mutans (EO and penicillin G) and E. faecalis (EO and ceftriaxone), whereas no effect was found against other bacteria. In the first case, there was a four-fold reduction in the MIC value; the MIC value decreased sixteen times when the EO was combined with ceftriaxone. Antagonistic interactions have not been reported in any of the papers analyzed in this work.
This body of evidence proves that plants of this family not only possess antibacterial activities but also can enhance the effects of antibiotics. The authors relate the potential antibiotic effect of EOs to the presence of monoterpenes and sesquiterpenes.

Relationship between the Chemical Composition of EOs and Their Antimicrobial Activity
The antimicrobial activity of EOs depends on their chemical composition and on the amount of the single components. Most EOs have a greater effect on Gram-positive bacteria than on Gram-negative bacteria. This behavior is attributed to the differences in the bacterial cell membrane composition. In Gram-positive bacteria, hydrophobic components easily penetrate the cell wall and act upon it as within the cytoplasm. Gram-negative bacteria have a peptidoglycan layer that is 2-3 nm thick, and an outer membrane (OM) lies outside the thin peptidoglycan layer and is firmly linked to it by Braun's lipoprotein embedded in the OM. This is composed of a double layer of phospholipids that is linked to the inner membrane by lipopolysaccharides (LPS). The peptidoglycan layer is covered by an OM that contains various proteins as well as LPS, which makes the bacteria more resistant to EOs and other natural extracts with antimicrobial activity [107,109].
The mechanism of action of EOs depends on their chemical composition, and their antimicrobial activity is not attributable to a single mechanism; this is widely described by Nazzaro et al. in their work [109]. The effect of the chemical constituents depends on their amount in the EOs, i.e., at low concentrations, they can interfere with enzymes involved in the production of energy and, at higher concentrations, they can denature proteins [109,110]. Examples of chemical components of EO tested experimentally are discussed below.
As previously described, β-caryophyllene, citral, 1,8-cineole, linalool, thymol, limonene, and carvone are volatile substances present in several EOs extracted from plants with recognized antimicrobial properties. These compounds presented antimicrobial activity. The possible mechanisms of action of these compounds are described in the following paragraphs.
Citral exhibited antimicrobial activity against pathogenic and food-spoilage bacteria such as E. coli O157:H7, Salmonella enterica serovar Typhimurium, Listeria monocytogenes, and Staphylococcus aureus [111,112]. This compund disrupts and penetrates the lipid structure of the cell wall of bacteria. It leads to protein denaturation and destruction of the cell membrane, followed by cytoplasmic leakage, cell lysis and death [113]. Citral was reported in the EO of L. alba (citral chemotype).
Linalool is one of the main components of some of these EOs (A. sellowii, L. alba) and it was previously reported to cause an increased permeability not only of the negatively charged membranes but also of fungal cells [114,115]. Because of the nature of their chemical structure, alcohols possess a strong binding affinity to different molecular structures, such as proteins or glycoproteins. Hence, they have great affinities for cell membranes and exhibit high potential to permeate cell walls, leading to the leakage of cytoplasmic material [116,117].
Thymol is one of the monoterpene phenols present in EOs from plants belonging to the Verbenaceae family (A. triphylla, L. graciliis, L. grandis, L. origanoides, L. sidoides). Its biological activities include antioxidant, anti-inflammatory, local anaesthetic, antinociceptive, cicatrizing, antiseptic activity, and especially antibacterial and antifungal properties [118]. Some authors [118,119] speculated that the antimicrobial effect of thymol might result, at least in part, from a perturbation of the lipid fraction of the bacterial plasma membrane, resulting in the leakage of intracellular materials. Xu et al. [120] confirmed it, because they demonstrated that this natural compound induces the permeabilization and depolarization of the cytoplasmic membrane. In addition, Chauhan & Kang [121] evaluated the antimicrobial properties and mechanism of action of thymol against S. typhimurium and showed the disruption of membrane integrity. They concluded that this is the main mechanism of action of thymol.
Germacrene-D is an organic compound belonging to the class sesquiterpenoid germacrane. The sesquiterpene hydrocarbon germacrene has five isomers, i.e., Germacrene A, B, C, D, and E. Germacrene D possesses antibacterial properties [122,123]. This sesquiterpene is present in several of the EOs studied, among which we can mention EOs of A. gratissima, L. camara, L. montevidensis, L. alba.
The EO of L. alba and its main components, such as citral and carvone, presented antibacterial and antibiofilm activities against S. aureus. The lowest MIC and MBC values were 0.5 mg/mL when L. alba EOs, citral, and carvone were used. The inhibition (100%) of S. aureus biofilm formation and the elimination of biofilm cells were confirmed. No elimination of biofilm cells was observed when carvone was used. Carvone is a monoterpene reported as one of the most effective antimicrobial agents present in several plants. Its main mechanism of action comprises the destabilization of the structure of phospholipids and the interaction with membrane proteins, and it acts as a proton exchanger reducing the pH gradient across the membrane [124].

Conclusions
Essential oils of South American plants from the Verbenaceae family contain as their main components monoterpenes and sesquiterpenes, such as thymol, β-caryophyllene, citral, 1,8-cineole, carvone, and limonene. The presence of these compounds, which increase or alter the permeability of bacterial membranes, could explain their antimicrobial action and their synergistic effect with antibiotics.
Pharmaceutical industries are in need of eco-friendly alternatives to drug molecules to treat infectious diseases. Thus, these EOs might be a prospective source of alternative antimicrobial agents and may play an important role in the discovery of new drugs against a wide range of pathogenic microorganisms in the near future.