Chemical composition, antimicrobial activity and synergistic effects with conventional antibiotics under clinical isolates by essential oil of Hymenaea rubriflora Ducke (FABACEAE)

Abstract This work aimed to investigate the chemical composition, antimicrobial activity, synergistic effect, and structure changes of the essential oil of Hymenaea rubriflora (EOHr). Forty-five constituents were identified in the essential oil, corresponding to 94.43% of the compounds present, being the main components E-Caryophyllene (36.72 ± 1.05%), Germacrene D (16.13 ± 0.31%), α-Humulene (6.06 ± 0.16%), β-elemene (5.61 ± 0.14%) and δ-Cadinene (3.76 ± 0.07%). Antimicrobial activity was evaluated, presenting antibacterial and antifungal activity with MIC ranging from 0.62 to 40 μL/mL. The essential oil had a synergistic effect when combined with gentamicin and fluconazole. Structural changes were also evaluated and it was possible to observe that EOHr action was related to changes in membrane permeability. The findings obtained here suggest that the use of the essential oil of H. rubriflora in the treatment of infectious diseases presents a potential for the future development of pharmaceutical products. Graphical Abstract


Introduction
Natural products have been playing an important role in the discovery of new antibacterial agents (Veras et al. 2020). Essential oils are produced in response to a stress condition suffered by the plant, for example a phytopathogenic infection (Chaturvedi et al. 2019). Its hydrophobic nature and complex chemical composition, give a low risk to the development of microbial resistance, since its action is attributed not only to a single specific compound, but may be related to the synergism between them due to the complementarity of different mechanisms, which hinders the microbial adaptations (Nafis et al. 2019).
Brazil has a huge plant diversity with several botanical genera, among them the Hymenaea (Fabaceae) being a genus with neotropical distribution, comprising approximately 16 species distributed in Central America, the Antilles and all South American countries, except Uruguay and Chile (Junior et al., 2019). Among various species of the Hymenaea genus, H. rubriflora Ducke known as "jatoba" and/or "red jatoba", has its leaves, stems and roots used in traditional medicine to make teas, infusions and mixtures for the treatment of various diseases such as lung weakness, asthma, anaemia, diarrhea, intestinal colic, cystitis, viral diseases, bacterial and fungal infections. However, to date, there are no reports on the chemical composition of the essential oil of this species, and its biological potential (Boniface et al. 2017).
In this context, this study aimed to investigate the first report of the chemical composition of essential oil of leaves from Hymenaea rubriflora Ducke, and evaluate its antimicrobial activity against twelve microorganisms, besides the potential synergistic effect with conventional antibiotics (gentamicin and fluconazole), and as well as its mechanism of action by observing the morphological alterations to pathogen structures that it caused viewed by scanning electron microscopy (SEM).

Chemical composition
The leaves of Hymenaea rubriflora Ducke were collected in countryside of Igarassu, in the state of Pernambuco, Brazil. The essential oil was obtained by hydrodistillation (HD) in a Clevenger apparatus, and subsequently analyzed regarding chemical composition in chromatography coupled to mass spectrometry (GC-MS) and flame ionization detector (CG-FID) (Veras et al. 2020). It was possible to obtain the essential oil with a yield of 0.069% (v/w). Forty-five compounds were detected, which represented 94.43% of the total composition. Among them, E-Caryophyllene (36.72 ± 1.05%) was defined as the major one, followed by Germacrene D (16.13 ± 0.31%), a-Humulene (6.06 ± 0.16%), b-elemene (5.61 ± 0.14%) and d-Cadinene (3.76 ± 0.07%) ( Table S1). This is the first report of the chemical composition of the essential oil of H. rubriflora, evidencing the novelty of this work. The essential oil of H. rubriflora Ducke presents a varied chemical composition, being mostly composed of sesquiterpene substances, which is a characteristic of essential oils (Perricone et al. 2015).

Antimicrobial activity
The essential oil presented effective antibacterial activity against strains of Bacillus subtills ATCC 9524, Staphylococcus aureus ATCC 29213, S. aureus UFPEDA 700, S. aureus UFPEDA 705, S. aureus UFPEDA 726, Klebsiella pneumoniae ATCC 29665 and K. pneumoniae CCBH 23940 KPC þ and Pseudomonas aeruginosa ATCC 27853 with MIC values ranging from 5 to 40 lL/mL, and MBC values ranging from 10 to 40 lL/mL, relationship between MBC/MIC of 2 to 4, and antifungal activity against strains of Candida albicans URM-6543, C. glabrata URM-6393, C. parapsilosis URM-6557 and C. tropicalis URM-6741 with MIC values ranging from 0.62 to 1.25 lL/mL (Table S2). As noted above, the difference between the antimicrobial activities by the essential oil is explained by the diversity in composition, and the interaction of each component in the target microorganism, as observed by Khoury et al. (2019).

Synergistic effects
The essential oil was investigated for possible synergistic effect combined with aminoglycoside gentamicin and with triazole fluconazole. It was observed that the EOHr presented potential synergic effect against bacterial resistance when combined with gentamicin ( Figure S1A) promoted a 2-fold reduction of MICs values in S. aureus UFPEDA 705 and K. pneumoniae CCBH 23940. It was possible to verify the 2-fold decrease in MICs values of fluconazole when combined with EOHr in the strains of C. tropicalis URM-6741 and C. glabrata URM-6393 ( Figure S1B). Resistance to antifungals and antibacterials such as fluconazole and gentamicin has become a clinical challenge for the treatment of infections caused by these drugs, are economically accessible and become the first choice in the treatment of infections caused by different species (Khoury et al. 2019). Several studies have investigated and proven possible synergistic interactions of essential oils with conventional antibiotics, and it is possible to reverse the resistance of microorganisms to antibiotics, which may become adjunctive to antibiotics (Dra et al. 2017).

Effects of essential oil on membrane integrity
The loss of UV absorbing material at 260 nm of the strains treated with MIC of EOHr for each microorganism is shown in Figure S2. These results suggest that EOHr is able to promote the increase in loss of absorbent material when compared to control, showing statistically significance in the strains of K. pneumoniae CCBH 23940, S. aureus UFPEDA 705, B. subtilis ATCC 9524 and C. glabrata URM 6393, K. pneumoniae ATCC 29665 and in S. aureus ATCC 29213. The increase in loss of UV absorbing material at 260 nm in the strain of C. glabrata URM-6393 treated with EOHr can be justified by the high percentage of sesquiterpenes in the chemical composition. Such effect was ascribed to cell membrane rupture (Shi et al. 2018).

Scanning electron microscopy
Scanning electron microscopy images of S. aureus 705, K. pneumoniae 23940, B. subtilis ATCC 9524 and C. glabrata URM-6393 cells submitted to treatment with MIC/2 of EOHr are shown in Figure S3. S. aureus cells, when treated with EOHr, were irregular, with surface depressions and consequent cell membrane cracks compared to the control ( Figures S3A and S3E). These results indicated that treatment of the essential oil resulted in damage to S. aureus. The K. pneumoniae cells treated with the essential oil revealed a severe damaging effect on the cell which presented irregular size, incomplete divisions and surface changes when exposed to EOHr ( Figure S3F). The cells of B. subtilis when treated with essential oil ( Figure S3G) changes were observed as: cell elongation with formation of filaments, extensive septa and cellular atrophy. Scanning electron microscopy of the Candida glabrata URM-6393 strain treated with essential oil was performed, cells showed morphological alterations including cells surface abnormalities. Sprouting structures ( Figure S3H) were observed in smaller amounts when compared to the control ( Figure S3D). Overall, electron micrographs have shown that essential oil of H. rubriflora at sub-inhibitory concentration promoted severe cellular changes, such as increased cell permeabilization, membrane integrity disturbance, septum-free cell elongation, irregularly wrinkled surfaces, incomplete divisions and filament formation, giving possible mechanisms of action of the essential oil on the microbial cells (Khoury et al. 2019).

Conclusion
The essential oil of H. rubriflora presented as major constituents E-Caryophyllene (36.72 ± 1.05%), Germacrene D (16.13 ± 0.31%), a-Humulene (6.06 ± 0.16%), b-elemene (5.61 ± 0.14%) and d-Cadinene (3.76 ± 0.07%). The essential oil showed antibacterial and antifungal activities, being a promising alternative for adjuvant in the therapy allied to aminoglycosides and azoles. Preliminary results showed that the mechanism of antimicrobial action of the essential oil may be related to permeabilization of the cells and disruption of the membrane integrity. It can be concluded that EOHr can be used as a natural source of antimicrobial agents to prevent economic loss by decreasing health risks from microbial pathogens such as bacteria resistant to various antibiotics, making it a candidate for drug development.

Disclosure statement
Authors declare that there is no conflict of interest.