Chemical Composition, Antioxidant, and Antibacterial Activities of Essential Oil of Atriplex semibaccata R.Br. Aerial Parts: First Assessment against Multidrug-Resistant Bacteria

: Atriplex semibaccata R.Br. is a perennial halophyte that has received much attention for studies of revegetation of marginal lands in arid and semi-arid environments. It was, recently, demonstrated that there are no risks in terms of contamination of essential oil (EO) from growing plant on such land. Interest in exploring the antibacterial and antioxidant potential of A. semibaccata EO has consequently been renewed. The objective of this study was to investigate the chemical composition, as well as the antioxidant and antibacterial activities of A. semibaccata EO. The antibacterial activity was evaluated against native (drug-sensitive) and multidrug-resistant (MDR) bacteria by testing the EO alone and in combination with conventional antibiotics. The chemical composition of EO was analyzed by gas chromatography/mass spectrometry, 52 chemical compounds were identiﬁed, and 2-Methoxy-4-vinyl phenol (48.9%), benzaldehyde (6.7%), and benzyl alcohol (6.3%) were found to be the main constituents of EO. Furthermore, the antioxidant activity was evaluated using a 2,2-diphenyl-1-picrylhydrazyl reducing–scavenging test. The EO from this species possessed high antioxidant activity (938.65 µ g TE/g EO). The antibacterial test demonstrated an inhibitory effect on six native and MDR bacterial strains. We found that Staphylococcus aureus (Gram+), Klebsiella pneumoniae (Gram − ), and Escherichia coli (Gram − ) were more sensitive than MDR strains, with an inhibition zone ranging from 11.16 mm to 12 mm. Moreover, the minimum inhibitory concentration ranged from 3.12 mg/mL to 6.25 mg/mL. The combination of gentamicin and EO revealed a high synergistic effect. The effect on S. aureus and K. pneumoniae showed lower fractional inhibitory concentration indices of 0.39 and 0.27, respectively. The results also revealed that A. semibaccata EO contained compounds with antibacterial potential against MDR bacteria, with antioxidant properties, and with a moderate synergistic effect in combination with gentamicin. The EO from A. semibaccata could be considered a new and potential source of natural antioxidant and antibacterial agents. These ﬁndings A. semibaccata


Introduction
The exploitation of marginal and saline land to produce useful biomass has attracted interest worldwide [1][2][3][4]. Plant species with valuable biomass in terms of bioenergy, biomaterials, and essential oil (EO) production may play a primary role in the revegetation of these lands, providing environmental and socioeconomic benefits [5][6][7]. Species Atriplex L. genus have been chiefly recommended for the restoration of saline and marginal lands [8].
Given its abundance in arid and semi-arid areas, its abiotic stress tolerance, and its suitability for use in reclamation, the Atriplex L. genus is interesting to explore in terms of the antibacterial potential of EO, especially in light of the worldwide spread of multidrugresistant (MDR) bacteria [15]. MDR bacteria pose an increasing hazard to public health worldwide [16], and bacteria continue to develop resistance to many of the currently available antibacterial drugs [17][18][19]. However, many plant species have not been screened for antibacterial activity of their EO against such bacteria. Moreover, an avenue that has not been widely explored involves utilizing new pharmaceutical products, which have original and multiple mechanisms of action, synergistically with current agents, which may be more effective against MDR bacteria [20,21]. Importantly, Lal et al. [22] and Zheljazkov et al. [23] demonstrated that EO extracted from vegetal crops grown in contaminated environments were free from the risk of heavy-metal contamination.
A. semibaccata is a perennial Amaranthaceae species [24], originally from Australia and introduced into several regions of the world as a drought-and salt-tolerant forage crop [25]. It became a naturalized plant in Morocco, distributed in the Saharan and middle Atlantic regions, including the Haouz area [25]. A. semibaccata is a xero-halophyte species, that tolerates moderate and high salinity (up to 15 dS/m) and considered a pioneer plant in clay and silty loam soils [26].
The current work was undertaken to identify the chemical composition of A. semibaccata EO, to evaluate its antioxidant and antibacterial activities against MDR bacteria, and finally, to explore the antibacterial synergistic effect of A. semibaccata EO and conventional antibiotics on MDR bacteria. As far as we know, the present novel research investigated the antibacterial activity of A. semibaccata OE against MDR bacteria. Moreover, no other prior studies have investigated the synergistic interaction between A. semibaccata EO and conventional antibiotics.

Plant Material and Essential Oil (EO) Extraction
In March 2019, the aerial biomass (2500 g) of several A. semibaccata plants was harvested from an experimental field located at phosphate mine overburdens in the Kettara region, Morocco (470 m above sea level; 31 • 51 36" N and 8 • 9 36" W). A specimen of A. semibaccata was deposited and conserved under the voucher specimen code MARK-13 000 at the Regional Herbarium "MARK" of the Faculty of Sciences Semlalia, University of Cadi Ayyad, Marrakech, Morocco.
Extraction of A. semibaccata EO was carried out four times (4 × 150 g) using the following procedure: The collected aerial biomass (2500 g) was initially air-dried at ≈25 • C for 5 days; thereafter, the dried biomass (1650 g) was subjected to hydrodistillation using a Clevenger-type apparatus for 4 h. The obtained EO was dried over anhydrous sodium sulphate and stored in darkness at 4 • C until use.

Gas Chromatography-Mass Spectrometric (GC-MS) Analysis
The EO was analyzed using a Trace GC-MS system from Thermo ScientificTM (Trace 1300 GC, USA), fitted with a TG-5MS column (30 m × 0.25 mm × 0.25 µm) and used in the electron-impact ionization mode. The temperatures of the injector and the detector were set at 230 and 250 • C, respectively, and the electron-impact ionization energy was 70 eV. For analysis, 1 µl of EO was injected in splitless mode into the GC-MS instrument, and helium gas was used as a carrier gas at a flow rate of 1 mL/min. The sample was pre-diluted in acetone at a 1:100 ratio, and the oven temperature was programmed to increase at a rate of 3 • C/min from 60 • C to 230 • C, which was maintained for 10 min. Finally, the chemical components were quantified by external standard method using calibration curves generated by running GC analysis of representative compounds. The antioxidant activity of the EO extracted from the aerial parts of A. semibaccata was assessed by a 2,2-diphenyl-1-picrylhydrazyl (DPPH) test [27], where 50 µl of the EO diluted at different concentrations in methanol was mixed with 2 mL of methanolic DPPH solution (60 µM). After 20 min of incubation at room temperature in darkness, the absorbance of the samples was measured at 517 nm. A blank containing the same amount of methanol and DPPH solution was used as a negative control, while butylated hydroxytoluene (BHT) and quercetin were used as positive controls. The radical-scavenging activity was calculated using the following formula: where A blank is the absorbance of the blank sample (control) and A sample is the absorbance of the EO test sample. The sample concentration providing 50% inhibition (IC50) was calculated by plotting the percentage of inhibition against the concentration of the EO sample (y = 116.73x − 0.1372; R 2 = 0.99). The analyses were performed in triplicate, and the results were expressed as the mean ± standard deviation (SD). In addition, the radicalscavenging activity was reported as microgram Trolox equivalents per gram of EO (µg TE/g EO).

Reducing-Power Assay
The EO reductive potential was evaluated by following the procedure of Oyaizu [28]. Briefly, 1 mL of different concentrations of samples (EO and control substance) was mixed with phosphate buffer (2.5 mL, 0.2 mM, pH 6.6) and potassium ferricyanide (2.5 mL, 1%). The mixture was then incubated at 50 • C for 20 min. Then, after incubation, 2.5 mL of trichloroacetic acid 10% was added to stop the reaction. The mixture was centrifuged at 650× g for 10 min. Finally, the supernatant (2.5 mL) was removed and mixed with 2.5 mL of distilled water and 0.5 mL of 0.1% ferric chloride (FeCl3), and the absorbance was measured at 700 nm. BHT and quercetin were used as positive controls.
The concentration of the sample providing an absorbance of 0.5 (i.e., IC50) was calculated from the graph of the absorbance at 700 nm against sample concentration, and the results were expressed as an average of triplicate measurements.

Antibacterial Screening
The examination of the antibacterial activity of the EO was evaluated using the agar disc-diffusion method as recommended by the Clinical and Laboratory Standards Institute (CLSI) guideline M07-A10 [29]. For this purpose, sterile and saline suspensions at 0.5 McFarland standards were prepared from overnight cultures of the respective bacteria. The bacterial suspension was then streaked on Mueller-Hinton agar plates using a sterile swab. Then, 10 µl of EO at a concentration of 896 mg/mL were applied to sterile filter paper discs (6 mm in diameter) and placed on the surface of the inoculated medium. The plates were maintained at 4 • C for 4 h to allow diffusion of the EO and then incubated at 37 • C for 24 h. Antibacterial activity was evaluated by measuring the diameter of the growth-inhibition zones after 24 h. Ceftriaxone (30 µg/disc), cefoxitin (30 µg/disc), and gentamicin (15 µg/disc) were used as potent antibiotics for testing MDR bacteria, according to CLSI guideline M02-A12 [30].

Determination of the Minimal Inhibitory Concentration (MIC)
The minimal inhibitory concentration (MIC) was determined using the microdilution broth method [31]. A two-fold serial dilution of EO was prepared in 4% dimethyl sulfoxide, and 100 µL of each dilution was added to micro-wells that were previously inoculated with 100 µL of bacterial suspension. The microplates were then incubated for 18-24 h at 37 • C. The MIC was defined as the lowest concentration without visible growth of the tested bacteria, and p-Iodonitrotetrazolium chloride ≥ 97% (Sigma-Aldrich) was used as a microbial growth indicator, while gentamicin was used as a positive control.

Determination of Minimal Bactericidal Concentration (MBC)
The minimal bactericidal concentration (MBC) was determined according to CLSI guideline M07-A10 [29]. In brief, 0.1 mL of the suspension from wells without apparent microbial growth after incubation during MIC tests was spread on Mueller-Hinton agar in Petri dishes. The Petri dishes were then incubated at 37 • C for 24 h. The lowest concentration of EO at which incubated bacteria were completely killed was taken as the MBC.

Synergistic Interaction between EO from A. Semibaccata and Conventional Antibacterials
The synergistic effect of A. semibaccata EO and the antibacterial agent gentamicin was assessed using a MIC microdilution [21]. This test was achieved using strains that are sensitive to the conventional antibiotic. MICs of antibacterial agents were determined in the presence of EO at a final concentration of MIC/4 for gentamicin. Briefly, 50 µL serial dilutions of gentamicin were added to microwells previously seeded with 100 µL of cell suspension at 108 colony-forming units/mL and containing 50 µL of EO at MIC/4. The microplates were incubated at 37 • C for 18-24 h.
The analysis of the effect of the combination of gentamicin and EO was calculated and expressed in terms of the fractional inhibitory concentration index (FIC I ) using the following formula [32]: To interpret FIC I , the system proposed by Didry et al. [32] was adopted; that is, total synergism was found when FIC I ≤ 0.5, partial synergism when 0.5 < FIC I ≤ 0.75, no effect when 0.75 < FIC I ≤ 2, and antagonism when FIC I > 2. The gain in antibacterial activity was also calculated and determined as the ratio of the MIC for gentamicin alone to the MIC for gentamicin in combination with EO.

EO Composition
Hydrodistillation of the aerial parts of A. semibaccata by the Clevenger-type apparatus yielded a dark green and strong-smelling EO, with a density of 0.9 g/mL, and a freezing point above −21 • C. In addition, the average yield was 0.09 ± 0.001% (w/w) based on dried weight.
The GC-MS of the EO resulted in the identification of 52 compounds, representing approximately 83.3% of the total oil ( Table 1). The main compound was 2-methoxy-4-vinylphenol at 48.9%, followed by benzaldehyde (6.8%), benzyl alcohol (6.3%), and o-xylene (2.1%). The chemical analysis of A. semibaccata EO revealed the major presence of 2-methoxy-4-vinylphenol, which, as far as we know, has never been found in EO from other plants of this genus. This compound is a phenolic derivative, exerting a potent anti-inflammatory effect, and it can block the growth of mammalian cells by arresting the cell cycle [33,34]. In another study, EOs from A. semibaccata and A. undulata (Moq.) D. Dietr. were found to have three compounds in common: 3-Hydroxy-beta-damascone, beta-ionone, and vanillin [35]. Boutaoui et al. [35] demonstrated that extracts from aerial parts of A. mollis Desf. contained vanillin, and Chouitah et al. [35] showed that the EO from A. lentiformis (Torr.) S. Wats. contained linalool and 2,3-pinanediol. We also found some of these compounds as minor components of the EO from A. semibaccata.

Antibacterial Activity
The antibacterial properties of A. semibaccata EO and conventional antibiotics were investigated against six pathogenic bacterial strains, including MDR strains (Figure 1 and Table 3). The findings disclosed that the EO of A. semibaccata had an antibacterial effect to different degrees on all the tested strains, including MDR strains, albeit to different degrees. The diameters of the inhibition zones lay between 11.16 ± 0.76 mm and 20.66 ± 0.57 mm, whereas the conventional antibiotics did not display any activity against the MDR strains.
On the basis of the results reported in Table 4 and Figure 2, the MIC and MBC values for A. semibaccata EO were in the range of 3.12 to 6.25 mg/mL. Native bacteria were found to be more sensitive than MDR bacteria, with an appropriate MIC of 3 mg/mL. Concerning the MDR bacteria, methicillin-resistant S. aureus and K. pneumoniae were inhibited at a MIC and an MBC of 6.25 mg/mL, while the EO repressed the growth of E. coli at an MIC value of 3.12 mg/mL. The inactivity of gentamicin against the MDR strains is explained by the resistance of these strains to this agent [45], taking into account that for sensitive strains to gentamicin, MICs start from 2µg/mL. The chemical architecture of the bacterial cell membrane is the main factor involved in its responding negatively or positively to antibacterial agents [45].
The present study demonstrates promising results since the MIC values were found to be equal to the MBC values, indicating a bactericidal effect on both native and methicillinresistant S. aureus and K. pneumoniae, and native E. coli (ATCC 25922).
According to Chambers and Deleo [45], and Garcia-Alvarez et al. [45], the resistance of methicillin-resistant S. aureus is essentially related to the production of an auxiliary penicillin-binding protein, PBP2a, which renders it resistant to all β-lactams, except for the novel class of cephalosporins. Previous findings suggested that an outbreak of infection with K. pneumoniae occurred as a result of the generation of the production of extendedspectrum β-lactamase (ESBL) [46]. ESBL plays the main role in increasing the antibacterial resistance of K. pneumoniae [47]. Despite the multitude of antibiotic types that have been developed, the molecular mechanisms of K. pneumoniae's resistance to antibacterial drugs remain unclear and need to be elucidated [48,49]. To the best of our knowledge, this is pioneering research that examined the antibacterial potential of A. semibaccata EO against MDR bacteria. Therefore, our results can only be compared and discussed regarding closely related species. Benzarti et al. [50] reported that A. semibaccata was previously tested as an antifungal agent. Moreover, according to Siddiqui et al. [13] and Ksouri et al. [14], numerous species of Atriplex L., such as A. hortensis, A. canescens  Table 3. Antibacterial activity of essential oil from Atriplex semibaccata R.Br. and antibiotics using the disc-diffusion method.  The antibacterial potency of A. semibaccata EO might be explained by the fact that its main compound, 2-methoxy-4-vinylphenol, has antibacterial potency [51,52]. Furthermore, the significant presence of other chemical components such as benzaldehyde and benzyl alcohol, also contributes to its antibacterial properties [53][54][55]. Benzaldehyde has been reported to have a bactericidal effect on human pathogens [56,57]. Moreover, benzyl alcohol is one of most frequently employed antibacterial preservatives in commercial peptide and protein products [55,58].

Synergistic Interactions between A. semibaccata EO and Conventional Antibiotics
Drug synergism between conventional antibacterial agents and plant EOs is a new approach to defeating the defense systems of microorganisms [21,59]. For this reason, our research attempted to explore potential interactions between EO of A. semibaccata and gentamicin as a conventional antibiotic.
The antibacterial effects of the EO with combined conventional antibiotics on selected pathogenic bacteria were explored by the checkboard method, and the results are presented in Table 5. The FIC and the FIC I were calculated to evaluate the synergistic activity of the EO in combination with gentamicin. The gain reported in the MIC of gentamicin in combination with A. semibaccata EO is also summarized in Table 5. Gentamicin exhibited a strong synergistic interaction with A. semibaccata EO, achieving a gain of four-fold for native strains of both S. aureus (FIC I = 0.39) and K. pneumonia (FIC I = 0.27; Table 5).  Results obtained here cannot be compared to other authors' findings because, as far as we are aware, no previous study has investigated the synergistic interaction between the EO from A. semibaccata and an antibiotic. The present study demonstrated that the interaction between the EO of A. semibaccata and a standard antibiotic (gentamicin) was notably effective using lower doses (MIC/4). EO of A. semibaccata therefore offers high potential for the development of further antibacterial agents for use in the treatment of certain diseases [60].
EOs have been found to act in different ways at multiple levels, and microorganisms have been found to be incapable of overcoming the antibacterial activity of EOs, unlike when they are treated with many conventional antibacterial, which have only one restricted site or mechanism of action [61,62]. Furthermore, numerous authors have demonstrated that the antibacterial activity of EOs in combination with other compounds is more effective than that of the individual constituents alone [21,[63][64][65]. These combinations reduced the minimum efficient dose of an antibiotic [66].

Conclusions
This study found that the EO obtained from the aerial parts of A. semibaccata had antioxidant and antibacterial activities against MDR bacteria. The results also confirm that the combination of EO and gentamicin, as a classic antibiotic, has a synergistic interaction against bacterial strains, despite not having clinically relevant effects. Furthermore, this EO was found to be rich in bioactive compounds, mainly, 2-methoxy-4-vinylphenol, and a naturally occurring phenolic compound with potent properties. However, future research on the chemical composition of EO of A. semibaccata should consider the potential effects of a multitude of parameters, given that it depends on geographical location, genetic factors, plant material, climate, soil, harvesting period, and method of storage and extraction. Although A. semibccata R.Br. is well adapted to arid and semi-arid climatic conditions, and the moderate antibacterial activities of its EO were demonstrated in vitro, future in vivo investigations are necessary to validate these findings, by testing the EO and the cytotoxicity of its major components at different concentrations on several cell lines to confirm its effectiveness and safety.  Acknowledgments: Thanks are due to E. Redouane (Ph.D. student) and R. Ait Babahmad for their contribution in the revision of this article, and K. Benrazzouk (Ph.D. student) for her valuable assistance in the protocol for essential-oil extraction.