COMPARATIVE STUDY OF THE ANTIBACTERIAL ACTIVITY OF CINNAMON AND ORIGAN ESSENTIAL OILS AND THEIR PRIMARYCOMPONENTS ON AVIAN Escherichia coli STRAINS.

and Oukessou Mohamed 1 . 1. Department of Biological and Pharmaceutical Sciences, Hassan II Agronomic and Veterinary Institute, BP 6202, 1010, Rabat, Morocco. 2. Department of Food and Nutrition Sciences, Agronomic and Veterinary Institute Hassan II, BP 6202, Rabat, Morocco. 3. Avian Pathology Unit, Department of Pathology and Veterinary Public Health, Agronomic and Veterinary Institute Hassan II, BP 6202, RabatInstitute, Rabat, Morocco. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

The emergence of bacterial resistance to the usual antimicrobial agents is nowadays a major challenge in therapeutics for both humans and animals. As a result, the search for new antimicrobial agents has become a necessity. In this context, a great interest was focused on naturally occurring substances with antibacterial activity. The present study consists of evaluating the antibacterial activity of two essential oils (EO) from two aromatic and medicinal plants commonly used in humans in Morocco, namely, oregano (origanum vulgare) and cinnamon (Cinnamomum cassia), and their primarycomponents, carvacrol and cinnamaldehyde respectively, against antibiotic-resistantEscherichia coli strains of avian origin. Extraction of the EO was carried out by hydrodistillation, and their chemical analysis was carried out by gas chromatography (GC).The cinnamon EO is mainly composed of cinnamaldehyde (nearly 90%), followed by betacaryophyllen (6.1%), cinnamyl acetate and eugenol with (2.75%) and (0.46%); carvacrol is the primary component of oregano EO (65.51%), followed by gamma-terpinene (11.49%), p-cymene (6.27%), thymol (4%) and borneol (3.58%). The aromatogram showed interesting antibacterial activity of EO and their active ingredients against the used bacterial strains. The diffusion method on agar medium showedthat EO of cinnamon was more active than oregano EO against E. colistrains, with mean inhibition zones of 25.8+4.5mm and 17+3.7 mm, respectively. The obtained MICs showed also that cinnamaldehyde was more potent than carvacrol, with average MICs of 0.039% and 0.106%, respectively.

ISSN: 2320-5407
Int. J. Adv. Res. 6(9), 373-381 374 Introduction:-During le few last decades, the use of antibiotic agents (AB) encountered two major constraints namely the emergence of antimicrobial resistance andthe strong consumer awareness on the health impact of drug residues in food of animal origin. In front of this situation, the discovery of new antibacterial agents has become more than indispensable. To be innovative and bypass aforementioned problems, the new generation of antibiotics should be safe for the consumer and not or less subjected to bacterial resistance. One of the most promising alternatives toreach such an objective would be the exploration of natural resources as they constitute, by their biodiversity, the largest reserve of bioactive substances. Among these alternatives, plant extracts including essential oils (EO) have a high potential to replace antibiotics.
Essential oils (EO) are aromatic and volatile oily liquids obtained from plant material. They are naturally formed in special cells or groups of cells found in leaves and stems (Oussalah and al., 2006). Although the antibacterial properties of EO have long been recognized, the recent interest in natural derived antimicrobials has led to renewed scientific interest in these substances. Indeed, EOs have many biological properties including antibacterialeffects, without development of resistance phenomenon, antioxidant activity and the stimulation of the immune and digestive processes (Bouhdid and al., 2009). Their antimicrobial activity has been shown in vitro by numerous studies (Smith-Palmer andal., 1998; Hammer andal., 1999; Dorman and Deans, 2000; Elgayyar andal.,2001), primarily against pathogenic bacteria such as Clostridium perfringens, Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Listeria monocytogenes and Yersinia enterocolotica (Dorman and Deans, 2000;Fabio andal., 2003). Antimicrobial activity of EO is particularly assigned phenols (such as carvacrol, thymol and eugenol), alcohols (such as linalool) and aldehydes (such as cinnamaldehyde). Thus, the antibacterial activity of an EO is determined byits level of these constituents (Bouhdid and al., 2009). The objective of the present study was to evaluate thein vitro antibacterial activity of two essential oils, namely, Origanum vulgare EO and Cinnamomum cassia OE and their major components, carvacrol and cinnamaldehyde, on Escherichia coli strains of avian origin resistant to Enrofloxacin and Florfenicol.

Bacterial strains
The antibacterial activity of essential oils and their active ingredients was evaluated on Escherichia coli reference strain (ATCC25922) and on 40 Escherichia coli strains of avian origin isolated and identified in the laboratory of Avian Pathology at the IAV Hassan II Institute. The isolation of E. coli was made from the lung, liver, heart, bone marrow of the broiler.

Methods:-Essential oil extraction
The apparatus used for the hydrodistillation of essential oils is of the Clevenger type (Clevenger, 1928). It consists of a balloon heater, a Pyrex glass flask where the dried plant is placed and distilled water, a vapor condensing column (refrigerant) and a Pyrex glass collector which also receives the distillation extracts. The essential oils obtained are stored in a refrigerator in a brown glass bottle sealed at 4 ° C and in the shade.

Determination of Antimicrobial Activity
The antibacterial activity of EO was evaluated using two different methods.

The Aromatogram
The aromatogram technique (diffusion technique in solid medium) is used to evaluate the sensitivity of bacteria to EO (Yashphe andal., 1979). Pure EOs are dispersed in 0.2% agar solution, to promote contact between germs and compounds. According to the method described by Remmal andal.,(1993a),a 10%initial emulsion is prepared by adding 100 μl of EO to 900 μl of a sterile aqueous solution containing 0.2% (w/v) of agar. Then sterile petri dishes (9 cm) containing 9 ml of Muller-Hinton medium were inoculated with the bacterial suspension of a density of 10 6 CFU/ml of medium; after solidification of the medium, 6 mm diameter sterile disks, made from the Whatmanpaper, were deposited on the surface of the medium and impregnated with 5 μl of the solution of EO, Carvacrol or Cinnamaldehyde. In the case of the combinationof two products, each solution was used at 2.5 μl. The petri dishes are then placed at a temperature of + 4 ° C. for about one hour, and then incubated at 37 ° C. for 18 to 24 hours. The sensitivity of the organisms tested with EO is characterized by the formation of a clear circle (zone of inhibition) around the disks containing these oils. The inhibitory effect of EO was evaluated by determining the diameter of the inhibition zone formed in mm (Matasyoh and al., 2007).

Macrodilution technique in a liquid medium
The minimum inhibitory concentration (MIC) of the EO was determined by the macrodilution method (Remmal andal., 1993). Briefly, a stock solution is obtained by dissolving 20μl of EO solution with 0.2% of agar in 2 ml of TSB, then serial dilutions of the obtained concentration(between 1% and 0.0039% (v/v))were carried out in tubes containing 1 ml of TSB broth. A seeded tube without EO was used as a positive control and, sometimes, a tube containing EO alone serves as a negative control. The different tubes are inoculated with 10 μl of the bacterial suspension with a density of 10 6 CFU/ml. The tubes containing different concentrations of EO and the positive control are incubated at the same time at 37 ° C for 18-24 hours.The minimum inhibitory concentration (MIC) is the lowest concentration of oil to which no bacterial growth is visible to the eye after incubation for 24 hours at 37 ° C (NCCLS, 1999).

Results And Discussion:-Essential oil chemical analysis Chemical composition of EO of cinnamon
The composition of cinnamon EO used in our study is given in Figure 1. This figure indicates that Cinnamaldehyde is the main component accounting for almost 90%, followed by beta-caryophyllen (6.1%), cinnamyl acetate (2.75%) and eugenol (0.46%). These results are somewhat different from those specified by the European Pharmacopoeia. Indeed, according to the European Pharmacopoeia (2011), levels of the cinnamaldehyde isup to 75%, and those of eugenoland β-caryophyllene are up to 7.5% and1 to 4% respectively.Theses differences might be ascribed to multiple factors such as extraction mode and analysis technique.    The EO of cinnamon and oregano are among the EO with the strongest antibacterial power (with thyme, savory and clove). The bactericidal activity of cinnamon EO has been described on various strains of Escherichia coli, with a power exceeding streptomycin (Senhaji and al., 2007). The antibacterial effect of oregano EO against E. coli has been reported by many studies (Ultee and al., 2002).

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The superiority of cinnamon EO compared to that of Oregano observed in our study agrees with the result observed by Bouhdid and al., (2012) has been confirmed in several studies, including the one conducted, while oregano has shown interesting antibacterial activity, particularly against strains of S. aureus. However, strains of Ps. Aeruginosa expressed some resistance to this oil. These strains were more sensitive to cinnamon EO which is predominantly composed of cinnamaldehyde (Inouye and al., 2001;Friedman and al., 2002). Our results also agree with those of Mithandal., (2014), commercial cinnamon (Cinnamomum cassia) and oregano (Origanum compactum) essential oils were tested by diffusion technique, against some pathogenic bacteria, cinnamon EO expressed an effect significant inhibitor and superior to that of Oregano, Listeria monocytogenes, Salmonella Typhimuriumand Escherichia coli (Mith and al., 2014). These promising antimicrobial effects can be attributed to the presence of major bioactive constituents, including cinnamaldehyde and carvacrol. Our results differ from those of Valero and Salmeron (2003), because among 11 essential oils tested against Bacillus cereus, Oregano EO was more active than Cinnamon EO (Valero and Salmeron, 2003).The essential oils of oregano and cinnamon are potential candidates for an application in the treatment of infections. These two essential oils act by increasing membrane permeability, inducing collapse of membrane potential and inhibiting respiratory activity, leading to a loss of cell viability (Bouhdid and al., 2012).

Antibacterial activity of the purecomponents (Cinnamaldehyde and Carvacrol) Activity on reference bacterial strains
The EO of cinnamon and its main active ingredient cinnamaldehyde showed a higherantibacterial activity than the EO of Oregano and carvacrol. Similarly, the activity of cinnamon EO was, on average, more potent than that of cinnamaldehyde (Table 1). The E. coli strain showed variable sensitivity to EO and pure components. It is more sensitive to cinnamon EO than it is to cinnamaldehyde, but more sensitive to carvacrol than oregano EO. However, the cinnamaldehyde and carvacrol combination showed greater inhibitorypower than cinnamaldehyde and carvacrol alone. The antibacterial activity of cinnamaldehyde has been mentioned in the work of Mith and al.,(2014) who have shown that this active principle has high antimicrobial activity against E.coli ATCC 35150; E. coli O157: H7 S0575; Salmonella typhimurium ATCC14028 and Pseudomonas fluorescens ATCC1352, whereas carvacrol, with the exception of P. florescence, had a lower activity. This result is consistent with our observation in the present study. However, according to another study, carvacrol had the strongest effect against Listeria monocytogenes, followed by thymol, eugenol, cinnamaldehyde and iso-eugenol (Yamazaki and al., 2004).

Avian strains Cinnamaldehyde + Carvacrol and their association
Ten avian strains E. coli resistant to enrofloxacinbut sensitive to colistin were tested to evaluate the inhibitory effect of cinnamaldehyde and carvacrol and their association. The results of the aromatogram are shown in Table 2.  1  20  16  30  2  22  20  40  3  30  20  40  4  28  30  28  5  32  20  24  6  32  20  24  7  28  20  26  8  32  16  36  9  36  22  30  10  38  14  32 378 Average ± SD 30±4 20±3 31±5 The pure components showed antimicrobial activity which resulted in varying zones of inhibition depending on the product. They ranged from 14mm to 30mm for carvacrol and from 20mm to 38mm for cinnamaldehyde. Corresponding respectively to means of 20±3mm and 30±4mm. For the cinnamaldehyde and carvacrol combination, the mean value was 31±5mm with variations of 24 to 40 mm depending on the strain. The results confirm the high antibacterial activity of cinnamaldehyde compared to carvacrol on E. coli strains.

Essential oils and theirmajor active ingredients
The results show that the highest inhibitory effect against avian E. coli strains was obtained with cinnamon products, with mean values of 25  The antibacterial activity of cinnamon EO was, on average, more potent than that of cinnamaldehyde ( Figure 4). This suggests that the minority compounds present in the EO have a significant effect. On the other hand, the figure 5, shows that carvacrol is more powerful than oregano EO. This could suggest that antimicrobial activity of the oregano EO is mainly due to carvacrol which represents only 65% in this EO in our study. This result might be also explained by the presence of antagonistic molecules in the EO.

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According to Kalemba and Kunicka (2003), the sensitivity of a microorganism to EO depends on the properties of the latter and the microorganism itself. In general, Gram-bacteria are more resistant than Gram + due to the structure of their outer membrane. Indeed, the outer membrane of Gram-is richer in lipopolysaccharides and proteins that make it more hydrophilic, and thus prevents hydrophobic terpenes from adhering to it. Nevertheless, some low molecular weight phenolic compounds such as thymol and carvacrol may adhere to these bacteria by attachment to membrane proteins and lipopolysaccharides through their functional groups and thereby reach the more vulnerable inner membrane (Dorman and Deans, 2000).

Minimal Inhibitory Concentration of active ingredients:-
MICs of cinnamaldehyde and carvacrol were calculated using ten E. coli strains of avian origin ( Figure 6). The average MIC values for each of the active ingredients are 0.039% and 0.106% for cinnamaldehyde and carvacrol respectively, which confirms the high inhibitory activity of cinnamaldehyde in comparison with carvacrol.

Conclusion:-
The cinnamon and oregano EOs and their major constituents have shown an important inhibitory effect on Escherichia coli strains of reference and of avian origin, with a remarkable antibacterial activity of cinnamon EO and its active ingredient, cinnamaldehyde, compared with oregano EO and its major constituent, carvacrol. The effect of the combination cinnamaldehyde and carvacrol was stronger than the activity of the two active ingredients tested separately on E. coli strains. Cinnamaldehyde Carvacrol