Chemical composition of Schinus areira essential oil and antimicrobial action against Staphylococcus aureus

Abstract The antimicrobial activities of plant extracts have formed the basis of many alternative medicines. In this context, the genus Schinus L. (Anacardiaceae), exhibits many traditional uses in medicine. However, a few studies on the antimicrobial properties of Schinus areira essential oils were conducted. The essential oil from S. areira leaves from Santiago del Estero was obtained by hydrodistillation and twenty-eight compounds were identified using CG-MS-EI spectrometry. The sesquiterpenoid alcohol 1-epi-cadinol was the major compound, followed by δ-cadinene, alloaromadendrene, β-pinene, β-caryophyllene, and γ-cadinene. The essential oil obtained exhibited antimicrobial activity against Staphylococcus aureus, showing a bacteriostatic activity at 64 µg/mL and bacteriolytic activity at 256 µg/mL; in contrast, non antibacterial effect was observed in Escherichia coli in the assayed conditions. The antibacterial activity was accompanied by significant changes in Zeta potential on the S. aureus surface. The data obtained suggest that the essential oil of S.areira leaves presents potential use in pharmaceutical industries.


Introduction
Schinus areira L. [syn. S. molle L. var. areira (L.) DC.] commonly known as 'aguaribay', pepper tree, or pink pepper, is a native species from Argentina and presents many traditional uses such as parasiticide, insecticide, vulnerary and topical disinfectant among others. However, only few studies have been conducted in order to screen the antimicrobial properties of S. areira essential oil (E.O.) (Martins et al. 2014;do Prado et al. 2019). Furthermore, according to our knowledge one study was conducted with S. areira E.O. from trees locates in Jujuy (extreme northwest of Argentina) (Celaya et al. 2014), and another one reported antifungal activity from a specimen located in the center of Argentina (Sampietro et al. 2014). Since chemical composition of essential oils is variable due to the influence of extrinsic conditions such as geographic origin; we aimed to evaluate the chemical composition and antimicrobial action of essential oil S. areira from Santiago del Estero which is located in North-West of Argentina, and compare this data with previous studies from S. areira E.O. from other regions of Argentina and South America. Also, biophysical studies were carried out to describe the interaction and the effect of the E.O.on the bacterial envelope and mimetic biological membranes.

Chemical composition of essential oil
The extraction yield of S. areira oil from leaves was 1.08% (v/w) and twenty-eight compounds were identified and quantified (Table S1; Figure S1). Monoterpenes and sesquiterpenes hydrocarbons were the most represented class of volatiles. The sesquiterpenoid alcohol 1-epi-cadinol was the mayor representative compound (34.6%), followed by d-cadinene (15.52%), alloaromadendrene (6.99%), b-pinene (bicyclic monoterpene, 5.85%), b-caryophyllene (5.11%), and c-cadinene (4.63%). Some studies of essential oil obtained from leaves of the Schinus genera collected in Europe, North Africa, America, even in Argentina, have shown different chemical composition (Gomes et al. 2013;Salem et al. 2013;Martins et al. 2014;Solis-Quispe et al. 2016). Moreover, the chemical constitution of E.O. from S. areira was described for samples collected in C ordoba (Argentina), where a-pinene, limonene and ocimenone were reported as the main compounds (Scrivanti et al. 2003). Besides, the E.O. chemical composition from leaves of two S. areira trees collected in Jujuy, Argentina, revealed predominantly monoterpenes as myrcene, limonene, a-phellandrene, b-phellandrene, sabinene and camphene (Celaya et al. 2014). The variability in the chemical composition in the genera Schinus was attributed to the influence of extrinsic conditions based on geographic origin (climatic and soil-growth conditions), and the effect of intra-specific differences (Solis-Quispe et al. 2016). Recently, EOs from 11 Brazilian populations of S. areira were extracted and four groups were formed by the Average Linkage cluster analysis based on their major chemical components: sabinene; aand b-pinene; a-cadinol; and myrcene (Gomes et al. 2013). The a-cadinol group presents a very similar composition thant it is reported here. On the other hand, b-pinene and epi-a-cadinol were the main compounds found in S. areira E.O. from leaves of an specimen collected in Botucatu (São Paulo, Brazil) (do Prado et al. 2019).

Antimicrobial properties
The antibacterial activity of E.O. against S. aureus and E. coli was determined by agar overlay bioautography. The S. areira E.O. showed antibacterial activity against S. aureus at 25 mg and the growth inhibition halos were proportional to the quantity of E.O tested ( Figure 2S). In contrast, no activity on E. coli was detected up to 100 mg of the E.O. (data not shown). These results are in good agreement with the fact that essential oils, in general, are more effective on Gram-positive than in Gram-negative bacteria, since the Gram positive cell wall is less resistant to the entrance of the hydrophobic components (Burt & Reinders 2003).

Determination of minimal inhibitory concentration
In order to complement bioautography data, minimal inhibitory concentration ( It has been reported that E.O. from leaves of Schinus spp.were effective against Gram-positive bacteria such as Bacillus subtilis, Staphylococcus epidermidis, Staphylococcus aureus, Enterococcus faecalis, and Gram-negative bacteria as Pseudomonas aeruginosa and Salmonella enteritidis (Rouibi et al. 2010;Martins et al. 2014). As it was pointed above, the difference between antimicrobial activities of different Schinus EOs origin is explained by the diversity in the composition as well as the concentration of each component in the E.O. and the target microorganism. Also its worth to mention, that even in the same plant, the part used influences the resulted composition of E.O.; for example, as it was observed a differential antibacterial activity between E.Os obtained from fruits and leaves which was related to differences in chemical composition (Celaya et al. 2014;Martins et al. 2014). In particular the EO analyzed by do Prado et al. (2019), also obtained from leaves, presents higher concentrations of monoterpenes. On the other hand, the E.O. obtained both from leaves and fruit described in Celaya et al. (2014). All this data reinforcing the importance of conduct more studies with local flora.

Interaction of S. areira E. O. with the bacterial surface
The interaction of antimicrobial agents with the bacterial envelope may involve electrostatic interactions that affect the bacterial zeta potential and cell permeability (Ferreyra Maillard et al. 2018;Tissera et al. 2019). In this sense, in order to contribute to the elucidation of the mechanism of action of S. areira E.O., we studied the zeta potential and CFU/mL after 1 h of incubation of S. aureus with different E.O. concentrations. In Figure 3S it can be observed that the addition of E.O. was able to induce changes on zeta potential values on S. aureus, indicating that some components of the E.O. would be able to directly interact with the bacterial envelope. Interestingly, an "umbral concentration" is observed below 200 mg/mL, where the E.O. has no effect on the bacterial charges after 1 h incubation. This threshold concentration seems to be close to the bactericidal concentration found, suggesting that at this point the changes on the bacterial envelope are irreversible after 24 h incubation but they are detectable as soon as 1 h incubation. When the viability curve was analyzed, a continuous drop on the CFU was observed as a function of the E.O. concentration tested, even when no changes on Zeta potential were observed. Since Zeta potential only senses the externally exposed charges, this could be explained by internalization of the active compounds after 1 h incubation, with no sufficient accumulation of the compound in the surface capable to induce a noticeable change on the surface charge.

Effect of S. areira E.O. on surface membrane potential in a biomimetic system
The degradation of the cell wall along with alterations of the membrane permeability has been described as a mechanism of action of E.O. In this context, zeta potential alterations of liposomes, that simulate bacterial membranes (DMPC: DMPG; 5:1) (Shireen et al. 2015), after S. areira E.O. addition were studied. Changes in the liposome zeta potential were observed as a function of the increasing concentration of S. areira E.O ( Figure 3S). Interestingly, in contrast to effects observed on whole S. aureus cells, changes in Zeta Potential on liposomes were significant at low concentrations, showing that there was no sharp threshold concentration ( Figure 3S). Also, in the case of model membranes, an increase in the net negative charge of the membrane was observed ( Figure 3S), while in the S. aureus potential measurements, the opposite effect was observed at high concentration of E.O. ( Figure 3S). Although more studies are necessary, this behaviour suggests that different components of the E.O. may directly interact with different components of the cell, like peptidoglycan and lipoteichoic acid in the case of whole-cell experiments and phospholipid in the case of model bacterial membrane.

Conclusions
The main composition of the essential oil of S.areira from northern provididence of Argentina (Santiago del Estero) was represented by monoterpenes and sesquiterpenes. Regarding its antibiotic activity, S. areira essential oil showed a MIC value against S. aureus of 64 lg/mL and a bactericidal activity at 254 lg/mL. In addition, biophysical studies using S. aureus and model membranes suggest a direct and differential interaction, where some components of S. areira essential oil seem to interact with the bacterial wall, others interact with the cell membrane. Beside further studies should be carried out in order to establish the role of each component of the E.O. in the observed antimicrobial activity, the fact that the E.O. exhibited a high antibacterial effect against S. aureus represents an important milestone for the future development of antimicrobial compounds from natural flora of North Argentina.

Disclosure statement
No potential conflict of interest was reported by the authors.