2.1. Chemical Profile of the Essential Oils
After extraction, the essential oils obtained from the leaves of Eugenia brasiliensis
Lam and Piper mosenii
C. DC. presented yields of 0.27% and 0.49%, respectively. A chemical analysis of these oils detected the presence of 20 chemical components in OEEb and 18 components in OEPm, as shown in Table 1
and Table 2
. Additionally, this analysis showed α-pinene and bicyclogermacrene as major constituents of OEPm, whereas α-muurolol was the main compound identified in OEEb (Figure 1
The composition of the essential oil of leaves of E. brasiliensis
Lam was analyzed by previous studies, which demonstrated the presence of several chemical components, such as spatulenol, α- and β-pinene, τ-cadinol, and α- and β-selinene [20
]. A study by Moreno et al. [22
], described the terpene 1,8-cineol as a major constituent present in the essential oil of this plant. However, this compound was found in lower quantity in the present study, indicating that the composition of the essential oil may vary according to the conditions of the sample used in each study. On the other hand, in the study by Fidyt et al. [23
], α-β-carrageenan, α-copaene, and pinene (α and β) were identified as major constituents, corroborating with the present study which described α-pinene as a major component of the essential oil.
Bernuci et al. [16
] described for the first time the composition of the essential oil of the species Piper mosenii
, mentioning the presence of compounds such as β-pinene, p
-cymene, α-thujene, (E
)-cariophyllene, bicyclogermacrene, γ-cadinene, trans
-aromadendrene, globulol, viridiflorol, and α-cadinol. It is worth noting that there are far fewer studies on this species than on E. brasiliensis
Differences in the composition and concentration of components of essential oils in studies with the same plant may be justified by variations on ecological and environment factors such as climate, relief, temperature, and soil type, as well as the part of the plant used in each case [24
]. According to Radünz et al. [25
], the accumulation of active principles can also vary according to the period of the year or due to genetic and physiological factors of the species, besides the age and the drying process of the leaves.
2.2. Antibacterial Activity by Direct Contact
An evaluation of the antibacterial activity of the essential oils of the two species demonstrated that OEPm was active against Staphylococcus aureus
, with a MIC of 512 μg/mL (Table 3
). Exposure to blue LED light did not modify the MICs of the treatments, indicating that, under the conditions of the present study, the blue light exhibited no modulating effect on the antibacterial activity of the essential oil obtained from Piper mosenii
On the other hand, the essential oil of Eugenia brasiliensis
Lam did not present clinically relevant antibacterial activity against strains of S. aureus
and E. coli
with MIC values ≥ 1024 µg/mL. These data differ from those of a study by Magina et al. [26
], in which an essential oil obtained from the leaves of this species presented moderate activity against E. coli
(MIC = 624.9 μg/mL) and strong activity against S. aureus
(MIC = 156.2 μg/mL) in tests performed by the microdilution method. These differences may be related to differences in the chemical composition of the products used in each study, because, in the essential oil obtained in the work of those authors, spatulenol (12.6%) and s-cadinol (8.7%) were the major constituents, whereas the essential oil obtained in the present study had α-muurolol (12.01%) as principal constituent.
Regarding the antibacterial activity of Piper mosenii
C.DC., it is emphasized that both standard and multi-resistant strains of Staphylococcus aureus
had their growth inhibited by the essential oil of this plant. These data are supported by a study using the diffusion disc method that showed that the Piper
species have antibacterial potential. It was demonstrated that the crude ethanolic extract, in addition to the hexane and chloroform fractions of the Piper mollicomum
leaves, showed antibacterial activity against standard strains of S. aureus
]. A study demonstrated the bioactivity of α-pinene, a major constituent of the essential oil of P. mosenii
C. DC., against strains of Staphylococcus aureus
, Salmonella pullorum
, and Klebsiella pneumoniae
as well as other bacterial species [28
]. Moreover, this compound exhibited significant activity against Mycobacterium tuberculosis
] and its positive enantiomer demonstrated to be active against Methicillin-resistant Staphylococcus aureus
(MRSA), Candida albicans
, and Cryptococcus neoformans
, demonstrating an antimicrobial effect that appears to be related to the chemical structure [30
It is hypothesized that bicyclogermacrene (the second major constituent of the oil) contributed to the antibacterial effect of P. mosenii C. DC. However, the isolated action of this against both standard and multi-resistant bacterial strains remains to be investigated.
Interestingly, the essential oils of P. mosenii
and E. brasiliensis
showed no activity against E. coli
. As Gram-negative bacteria, E. coli
has a cell wall rich in lipopolysaccharides that inhibit the entry of several antimicrobial substances. Therefore, these microorganisms are frequently less sensitive to the action of essential oils [31
2.3. Modulating Effect of Essential Oils in Association with Blue LED Light on the Activity of Aminoglycosides
The antibacterial effect of the blue LED light is well known in the literature [18
]. However, against both bacterial strains assayed, the blue LED light alone demonstrated a MIC ≤ 1024 μg/mL. In the modulation tests, association of OEEb with amikacin and gentamicin presented a synergistic antibacterial effect against the multi-resistant strain of Escherichia coli
). The effect of this association was further enhanced was by exposure to blue LED light. In the tests with the multi-resistant strain of Staphylococcus aureus
, association with OEEb or blue LED light increased the activity of amikacin. However, the simultaneous association of OEEb with blue LED light and the antibiotics did not present a significant antibacterial effect.
In the test with OEPm, association of this substance with amikacin did not affect the activity of this antibiotic against E. coli
). However, when this combination was exposed to blue LED light, the antibacterial effect was potentiated, indicating that light exposure stimulated synergistic interactions between the treatments. Interestingly, in the tests with gentamicin, blue light, and OEPm, all combinations showed synergism against this microorganism. Considering these combinations against S. aureus
, all conditions presented synergism with amikacin, but no modulation was observed with gentamicin.
These results indicate that blue LED light act as a modulator of the antibiotic activity of aminoglycosides, especially in the presence of the essential oils obtained from the plants under study. It is worth noting that the modulating effect of blue light associated with essential oils was stronger against E. coli, which resisted the modulating effect of OEPm in the absence of light exposure.
The results obtained in the present study corroborate those described by Pereira et al. [32
]. Through the gaseous contact method, the authors observed a synergistic effect from the association of blue LED light with the essential oil of Eugenia jambolana
and antibiotics against strains of Escherichia coli
and Staphylococcus aureus
. According to the authors, investigation of the combined effect of essential oils and LED lights may represent a key step in development of novel therapies against infections caused by resistant bacteria.
Promising results were also obtained by Matias et al. [33
] studying a combination of blue LED light, antibiotics, and the essential oil of Cordia verbenacea
DC. by the gaseous contact method. In the presence of ciprofloxacin and norfloxacin, the association of the oil and blue light presented a synergistic effect against E. coli
and S. aureus
. However, in the presence of gentamicin and amikacin, the same combination was effective only against S. aureus
. These results differ partially of those obtained in the present study, which may have occurred because of differences on the methodologies or in the chemical constitution of the essential oils analyzed.
According to Caffarel-Salvador et al. [34
], the use of photodynamic therapy is a new form of antimicrobial therapy, which is already used for treatment of cancerous skin lesions and has been efficiently evaluated in the treatment of infections. This is a low-cost method that provides a biophysical and biochemical rebalancing in the treatment of diseases, besides being non-invasive [35
In terms of mechanism, phototherapy can cause activation of chemical compounds, as previously reported in a study by Brito et al. [36
]. This study demonstrated that inactive compounds present in the skin of the amphibian Rhinella jimi
acquired an antimicrobial activity after association with ultraviolet light.
The blue LED light can act by eliminating both Gram-positive and Gram-negative bacteria by inducing oxidative stress [32
]. This effect is triggered when the light is associated with chemical compounds found in medicinal plants that act as photosensitizers. Thus, the compounds are excited, which results in the formation of intracellular reactive oxygen species (ROS), causing oxidation of essential constituents of the bacterial cells with consequent death of these microorganisms [37
Therefore, it is suggested that components of the essential oils used in the present study may act as photosensitizing substances, because they had the antibacterial effect improved in the presence of blue LED light, modulating the activity of aminoglycosides and consequently reducing the MIC of these antibiotics. This phenomenon has great clinical relevance, because this type of therapy may reduce the dose of antibiotics required to treat infections, minimizing their side effects [39
]. Furthermore, association of LED light and essential oils can be used to improve the effectiveness of conventional antibiotics against resistant bacteria.