Chemical Composition and Antimicrobial Activity of the Essential Oils of Two Aromatic Plants Cultivated in Morocco ( Cinnamomum cassia and Origanum compactum )

The present study aims to evaluate the antibacterial properties of natural products according to a pharmacodynamic approach in order to propose them as alternatives to synthetic products. Two essential oils ( Cinnamomum cassia and Origanum compactum ) were the subject of the chemical and biological study. First, we evaluated the sensitivity of the strains of avian Salmonella to the main antibiotics used and then to the chromatographic analysis of the composition of the two essential oils (EO); ﬁnally, we proceeded to the in vitro evaluation of the antibacterial activities of these EO (alone and in combination with antibiotics). The results obtained showed that carvacrol (35.2%), followed by c -terpinene (20.1%), was the main constituent of the essential oil of O. compactum while cinnamaldehyde (69.1%) represents the major component of the essential oil of C. cassia . The antibioresistance proﬁle of the Salmonella tested showed resistance to ampicillin (35%) and oxytetracycline (41.3%). Active products extracted from the essential oils studied showed antibacterial activity against Salmonella strains. C. cassia products were shown to be more active for Salmonella enteritidis (average inhibition diameter: 16.3 mm) and for Salmonella gallinarum (average inhibition diameter: 27.7 mm). The best synergistic activity with antibiotics has been obtained with the essential oil of C. cassia and its active product cinnamaldehyde. The minimum inhibitory concentration (MIC) of cinnamaldehyde is the lowest (0.05%). The results prove the presence of an antibacterial activity and a synergistic eﬀect of two essential oils studied with the main antibiotics.


Background
Salmonellosis is the most common foodborne bacterial disease worldwide. e bacteria responsible belongs to the family of Enterobacteriaceae and the genus Salmonella and is a Gram negative, optional anaerobic, and most often mobile [1][2][3].
Currently, there are approximately 2610 Salmonella serotypes determined by their O and H antigenic structures and classified according to the Kauffmann-White classification updated annually [4].
Salmonella is a predominant cause of food infections. One study estimated that they were responsible for 93.8 million human cases of gastroenteritis and 155,000 deaths worldwide every year [5].
Furthermore, according to the European Food Safety Authority (EFSA) and the European Center for Disease Prevention and Control (ECDC), salmonellosis was the second most frequently reported gastrointestinal infection for people in the European Union in 2018 (91,857 cases), after campylobacteriosis (246,571 cases). https://www.efsa. europa.eu/fr/news/salmonella-most-common-causefoodborne-outbreaks-european-union. e misuse of antimicrobial agents has led to the emergence of resistant bacteria. is resistance to antibiotics represents a serious problem to human health [6]. e majority of antibiotic resistance is due to the genes that could be expressed in any bacteria. is notion is illustrated by the term "resistome" which defines the set of antibiotic resistance genes that can be found in environmental bacteria as well as in pathogenic bacteria [7].
Salmonella resistance to commonly used antimicrobials (such as tetracyclines, sulfonamides, ampicillin, and fluoroquinolones) has been detected in humans and also in poultry [8].
e kentucky and infantis Salmonella serotypes are of concern because they show a high level of resistance to ciprofloxacin as well as multiresistance to several other drugs [9]. Multiresistance is particularly observed for the strains of the Salmonella typhimurium serotype [10].
Alongside prevention measures, scientific research has oriented towards the development of new molecules not or hardly subject to the phenomenon of resistance and towards the discovery of products capable, by synergistic effect, of restoring sensitivity to already existing antibiotics.
In addition, in order to preserve the health of the consumer and to delay the wear of antibacterials without compromising the production performance of the chicken, chemicals based on extracts of aromatic plants can constitute a complete or partial alternative to the usual antibiotics [14,15].
However, as with all products used for human or animal health, rational use of natural products must be done on the basis of pharmacological, toxicological, and clinical studies.
is work aims, on the one hand, to contribute to the study of the resistance to antibiotics of Salmonella of the animal origin, to evaluate the antibacterial properties of essential oils of cinnamon (C. cassia) and oregano (O. compactum), as well as their main active components (cinnamaldehyde and carvacrol), and, on the other hand, to study their possible interactions with usual antibiotics.  (Figures 1(a) and 1(b)) were collected in 2018, at the Rabat region. e samples obtained were kept at 4°C until the time of analysis. e extraction of essential oils from the samples was carried out at the laboratory of the Department of Food Sciences of the Agronomic and Veterinary Institute (IAV Hassan II). e reference cinnamaldehyde (Sigma-Aldrich, 99%) and carvacrol (Sigma-Aldrich, >98%) were provided by the same department.

Salmonella.
e bacterial strains tested were isolated from autopsies of laying hens from different regions of Morocco.
e samples were taken and identified at the Avian Pathology Unit of IAV Hassan II by Nassik et al. [18].
Isolated bacteria were conserved at +4°C and kept alive by continuous subcultures on the agar trypticase soya (TSA) medium. Twenty one (21) strains were tested: ten (10) strains of Salmonella enteritidis, ten (10) strains of Salmonella gallinarum (sensitive to florfenicol and resistant to oxytetracycline and amoxicillin), and one reference strain of Salmonella (CTP 8059) provided by the Pasteur Institute of Casablanca/Morocco.

Extraction of Essential Oils.
e essential oils were extracted from the aerial parts of O. compactum and the bark of C. cassia, by hydrodistillation for three hours using the Clevenger-type apparatus. e distilled essential oils had been dehydrated with anhydrous sodium sulphate and stored at 4°C in dark, airtight bottles [19]. e oil yield was express as a percentage and calculated according to the following formula: where Rdt EO is the yield in the essential oil (%), M EO is the mass of the essential oil (g), and M V is the mass of the dry plant material (g).

Chemical Analysis of Essential Oils.
A gas chromatograph (CLARUS 580) mounted on a mass spectrometer (Clarus SQ 8S) and operating in the positive electronic impact mode (70 ev) and a VB-5 column (5% phenyl and 95% methyl polysiloxane), 30 m long, 0.25 mm internal diameter, and 0.25 mm thick, were used. e temperatures of the injector and the detector were 250 and 300°C. e gas used is helium at a flow rate of 1 ml/ min. e program temperature is from 50 to 280°C with a gradient of 8.1°C/min. Component identification was done using the combination of the NIST-MS Research Program and the PubChem database (https://pubchem.ncbi.nlm.nih. gov/).

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Journal of Chemistry

Antibiogram.
e antibiogram was carried out by the method of diffusion on the Mueller-Hinton agar medium. Some colonies are taken and diluted in sterile physiological water (dilution of 1/10 on a scale of 0.5 MacFarland). e antibiotic discs are deposited separately, and then the dishes are incubated at 37°C for 24 hours.

Evaluation of Antibacterial Activity.
e antibacterial activity of the essential oil was determined by the diffusion method on agar (Muller-Hinton) [20]. Two controls were performed: a negative control with 10 μl of sterile distilled water in the presence of 10% DMSO and a disk representing one of the antibiotics as a positive control. e dishes are incubated at 37°C for 18 to 24 hours. Next, the diameter of inhibition is measured in millimeters.

Determination of Minimum Inhibitory Concentration.
e minimum inhibitory concentration (MIC) was determined by the microdilution technique on a 96-well roundbottomed microplate [21], using resazurin as an indicator of viability [22]. A series of half dilutions of the essential oil (1/ 2, 1/4, 1/8, and up to 1/512) were prepared at a concentration range from 1% to 0.00195%, with the concentration of the stock solution of each oil being 1 μl/100 μl. e dilution method consists of preparing a series of wells containing tryptic soy broth (TSB). e wells are filled with 69 μl of TSB. en, 1 μl of essential oil is added to the first one and then a dilution is made. en, each well is inoculated with 25 μl of the bacterial suspension. e wells used as a negative control do not contain essential oil, and those used as a positive control contain essential oil without bacterial suspension. e microplates were incubated at 37°C for 18-24 hours. For MIC revelation, 5 μl of resazurin 0.01% (w/v) was added to each well. e MIC was defined as the lowest concentration of oil that did not produce a color change in the resazurin and corresponds to the absence of bacterial growth.

Determination of Minimum Bactericidal Concentration (MBC).
e MBC is calculated using 10 μl of wells, which have not shown bacterial growth on a solid medium after seeding and incubation at 37°C for 24-48 h. e MBC is considered the minimum concentration of essential oil for which no bacterial development is observed.

Interactions between Essential Oils, eir Majority Compounds, and Antibiotics.
e association of essential oils and their majority compounds with antibiotics was evaluated by the method of diffusion by discs in the solid medium recommended by Halawani and Toroglu [23,24]. is involves comparing the inhibitory activity on a strain of Salmonella using three discs: the first is an antibiotic disc (ATB), the second is a sterile neutral disc soaked in 5 μl of essential oil (EO) or the majority compound (PA) to be tested, and the third is an antibiotic disc soaked with 5 μl of the same essential oil or of its majority compound to be tested.
After incubation, the reading of three inhibition diameters in mm is carried out using a ruler. e data were analyzed according to the following three possibilities: A: indifference: the zones of inhibition of EO/PA alone, ATB alone, and the association EO + ATB remain unchanged B: antagonism: the inhibition zone of the EO + ATB association is less important than that of the EO/PA alone and the ATB alone C: synergy: the inhibition zone of the EO + ATB association is more important than that of the EO/PA alone and the ATB alone e method of analysis of variance (ANOVA) was used to test the effects of factors on the variables measured (diameter and percentage %). If we reject the null hypothesis of equality of means, we use Duncan's multiple comparison tests of means to form homogeneous groups of essential oils and active ingredients that do not differ between them. For the variables in percentage (MIC and MBC), we transformed them with the angular transformation before performing the ANOVA. ese analyzes were carried out using the SAS software (SAS, 2006).

Essential Oil Yield.
e essential oil of the bark of the cinnamon of China (C. cassia) is a yellowish liquid with a strong and characteristic odor with a yield of 1.18% (w/w). Journal of Chemistry e essential oil of O. compactum was calculated based on the dry plant matter of the aerial part of the plant and had a yield of 1.6%.
e number of components identified in the essential oil of C. cassia in our study is much smaller than that reported in some previous studies [25]. Figure 3 represents the percentages of resistance of the strains of Salmonella tested to antibiotics. Erythromycin has shown the maximum resistance (100%), which shows that Salmonella are naturally resistant to this antibiotic [26]. e resistance of the strains studied to oxytetracycline was 41.28% and 35% to amoxicillin, while enrofloxacin and trimethoprim-sulfamethoxazole had a resistance percentage of ,approximately, 23 and 13%, respectively. iamphenicol showed 9% resistance. However, colistin (25 and 50 μg) showed a resistance of 5% as well as florfenicol (5%).

Evaluation of Antibacterial Activity by the Disc Diffusion
Method. Aromatograms were performed first on a reference strain (Salmonella CTP 8059) and then on strains of Salmonella enteritidis and Salmonella gallinarum of the avian origin. e results are expressed by the diameters of the inhibition zones. According to Meena and Sethi and Ela et al. [27,28], susceptibility to an essential oil was classified according to inhibition zone diameters as follows: (i) EO strongly inhibitory: diameter of the inhibition zone >28 mm (ii) EO moderately inhibitory: diameter of the zone between 16 and 28 mm (iii) EO slightly inhibitory: diameter of the zone varies between 10 and 16 mm (iv) EO noninhibiting: inhibition diameter is less than 10 mm

Reference Strain.
e results of the antibacterial activity of essential oils and majority compounds were studied on the reference strain (Table 3). e null hypothesis (H 0 ) assumes that the mean inhibition diameters of the essential oils and majority compounds are equal. Since the probability value (p value) is less than 0.05, H 0 is rejected and the mean inhibition diameters are different.
Based on the results of Duncan's test, three different groups of essential oils were identified: (i) e 1 st group composed only of cinnamaldehyde, which gave the best average diameter of inhibition (30.3 mm) (ii) e 2 nd group is composed of the essential oil of C.
cassia and carvacrol with average inhibition diameters of 25 and 24.7 mm (iii) e 3 rd group is composed only of the O. compactum which gave the smallest diameter (21 mm) e essential oil of C. cassia (mean inhibition diameter 25 ± 2 mm) and cinnamaldehyde (mean inhibition diameter 30.0 ± 1.5 mm) (p < 0.005) showed a higher antibacterial activity than the essential oil of O. compactum (mean inhibition diameter 21 ± 2 mm) (p < 0.005) and carvacrol (mean inhibition diameter 24.6 ± 1.5 mm).
is indicates that the bacterial strain is more sensitive to C. cassia essential oil and cinnamaldehyde than to O. compactum essential oil and carvacrol.

Wild Strains of Avian Origin.
e null hypothesis H 0 means that the average diameters of inhibition of essential oils and majority compounds are equal and there is no interaction between strains and essential oils. If the probability value (p value) is less than 0.05, H 0 is rejected and the mean inhibition diameters are different.
Initial qualitative tests of essential oils and their majority compounds on the strains studied, using the agar diffusion method, resulted in zones of inhibition (Table 4). e ANOVA indicates that there are highly significant differences between the two strains (F value � 265.73 and p  e essential oil of C. cassia presents, on average, the highest inhibitory activity on both strains (average inhibition diameter 22 mm) compared to the essential oil of O. compactum and carvacrol (average inhibition diameter 17.7 and 17.3 mm). However, this activity varies from strain to strain. Indeed, Salmonella gallinarum is more sensitive to the essential oil of C. cassia (average diameter of inhibition 27.7 mm) than the strain of Salmonella enteritidis (average diameter of inhibition 16.3 mm). e activity of the essential oil of C. cassia (average inhibition diameter 22 mm) is, on average, more powerful than that of cinnamaldehyde (average inhibition diameter 18.9 mm).
However, carvacrol seems to be more active (inhibition diameter 21.4 mm) than the essential oil of O. compactum (inhibition diameter 17.7 mm) for Salmonella gallinarum which can be justified by its low content in the composition of the essential oil (35.2%) or the presence of carvacrol antagonistic compounds in the essential oil of O. compactum [29].

Determination of Minimum Inhibitory and Bactericidal
Concentrations. Table 5 shows the results of the minimum inhibitory concentrations (MIC) and minimum bactericidal     According to the ANOVA results, there are significant differences only between the essential oils for the two parameters measured (MIC and MBC) (F value � 10.45 and p value � 0.0005; F value � 22.55 and p value <0.0001), whereas there is no significant difference between the two strains and no significant interaction between the strains and the essential oils.
In a liquid medium, the essential oil of C. cassia was, on average, the most effective, and its effect was also very stable (MIC 0.018% and MBC 0.026%). e essential oil of O. compactum was less effective than that of C. cassia (MIC 0.114% and MBC 0.167%). Similarly, cinnamaldehyde presented MICs of 0.04 and 0.05%, thus showing a strong antibacterial activity on the strains studied, thus translating a better antibacterial activity of the essential oil of C. cassia, which can be explained by the combined effect of cinnamaldehyde (69.15%) and other minority compounds [30]. e results obtained for the MICs are for the majority in concordance with the diameters of the inhibition zones observed in the agar diffusion test. On the other hand, all the products tested showed bactericidal activity, and the values of MBCs were similar or almost similar to those of MIC (with mean MIC of 0.076% and mean MBC of 0.10%). C. cassia exhibited the highest inhibitory and bactericidal activity against both strains.

Interaction of Essential Oils-Antibiotics.
We have associated each essential oil and each active principle with different antibiotics to which the Salmonella strain exhibits resistance, namely, oxytetracycline and amoxicillin, to qualitatively determine the possible interactions between essential oils as well as their active principles and antibiotics (Table 6).
According to the ANOVA results, there are significant differences between bacterial strains (F value � 976.82 and p value <0.0001), products (F value � 223.45 and p value <0.0001), and products used (F value � 76.05 and p value <0.0001). Similarly, all interactions are significant except that between the strains and the products used (F value � 0.60 and p value � 0.5787). e analysis of the data obtained shows that the association of the essential oil of O. compactum with oxytetracycline gave synergistic interactions (18 mm) compared to that of the essential oil alone (14.5 mm) and the antibiotic alone on both strains of Salmonella, while a slight antagonistic effect was obtained with amoxicillin (9 mm) compared to the essential oil alone (14.5 and 22 mm). Table 6 shows an antagonistic interaction when combining carvacrol with amoxicillin (15.75 mm); however, synergy is achieved with oxytetracycline (20 mm). e results show a synergistic interaction of the essential oil of C. cassia (23 and 22.75 mm) and cinnamaldehyde (21.5 and 20 mm) with all the antibiotics tested.
From the results cited above, we found that the essential oil of C. cassia and cinnamaldehyde were the most active in combination with antibiotics, and this result shows a synergistic effect.

Discussion
e study of the chemical composition of essential oils has shown that the majority constituents of the essential oil of O. compactum are carvacrol (35.17%), followed by c-terpinene (20.09%), thymol (17.55%), and o-cymene (13.67%), accounting for 86.48% of the oil. ese results are in agreement with those obtained in the study by Ben-Hammou et al. which showed that the most abundant compound in the chemical composition of this essential oil is carvacrol (36.31%), followed by thymol (16.88%) and p-cymene (9.21%) [31].
With regard to amoxicillin, 35% of the strains were resistant, which is a high result when compared to the percentage found by Ziyate et al. [38] which was 21%.
Almost 23% of the strains are resistant to enrofloxacin which is lower than the percentage found by Antunes et al. who reported that 75% of the strains isolated in Porto (Portugal) are resistant to one antibiotic or more and in particular to enrofloxacin [41].
Approximately, 13% of strains were resistant to trimethoprim-sulfamethoxazole, a result which remains low when compared to the rates reported by Gad and Hafez which is 59% in Germany [42]. For thiamphenicol, 9% of the strains are resistant which is due to the fact that it is no longer allowed in poultry.
A resistance of 5% to colistin (25 and 50 μg) was found. According to Humbert and Salvat, colistin remains among the sufficient number of antibiotics active on Salmonella [43]. And, a 5% resistance to florfenicol could be explained by the fact that this molecule is not commonly used in poultry.
e aromatogram study showed that the essential oils and active products tested have antibacterial activity against the reference strain and the bacterial strains studied. C. cassia's products have been shown as the most active.
is study revealed that S. gallinarum is more sensitive to the essential oil of C. cassia (27.65 mm) than the strain of S. enteritidis (16.25 mm). However, carvacrol seems to be more active (21.4 mm) than the essential oil of O. compactum (17.7 mm). e majority compounds are most often responsible for the antibacterial activity observed [44,45]. Nevertheless, other studies show that in addition to major compounds, minority compounds play an important role in the antibacterial activity of the essential oil [46]. On the contrary, the sensitivity of a bacterium to essential oils depends on the properties of the microorganism itself [44,47].  e results of the minimum inhibitory concentration showed that the MIC values vary depending on the strain and on the natural product used, and the MIC of cinnamaldehyde was the lowest when compared to the strains tested (0.04 and 0.05%). ese results are in agreement with those obtained in a previous work on Gram bacteria [48]. Nevertheless, the MIC values found remain higher than those obtained in our study, reflecting a better antibacterial activity of the essential oil of C. cassia [49,50]. e results of the interaction of essential oils and antibiotics also demonstrated the potentiating effect of essential oils and their majority compounds on the activity of the tested antibiotics. e best synergistic activity with antibiotics was obtained with the essential oil of C. cassia (23 and 22.75 mm) and its active product cinnamaldehyde (21.5 and 20 mm). Our results are, on the whole, comparable to those reported for O. compactum, C. cassia, and their active principles [51]. e association of essential oils with antibiotics can be used to increase the antimicrobial spectrum and reduce and minimize the side effects of the antibiotic [52].

Conclusions
e essential oil of oregano and its majority component, carvacrol, and, above all, the essential oil of cinnamon and its majority component, cinnamaldehyde, are active in vitro against the reference strain (Salmonella CTP 8059) and the strains of Salmonella enteritidis and Salmonella gallinarum. e lowest minimum inhibitory concentration (MIC) was obtained with cinnamaldehyde (0.05% (v/v)) against Salmonella enteritidis and (0.04% (v/v)) against Salmonella gallinarum compared with that of carvacrol (0.014% and 0.01% (v)) respectively, for the two bacterial strains. e minimum bactericidal concentrations (MBCs) of the four products were very close to their respective MICs, indicating that the products studied are rather bactericidal. e association of essential oils with antibiotics used in avian medicine indicates synergies, with the exception of the essential oil of O. compactum and carvacrol, which lend themselves much less to such interaction with amoxicillin. It should be noted that the resistance profile of strains isolated from poultry varies according to the antibiotic family and is comparable to the rates reported in other countries.

Data Availability
All the data generated or analyzed during this study are included within the article.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this article.