MATERIALS AND METHODS

Extraction of essential oil: The bark of Cinnamomum aromaticum was purchased from a specialized store in Tlemcen (Algeria). The essential oil was extracted by hydrodistillation process by mixing 50 g of crushed Cinnamomum aromaticum in 500 mL of distilled water for 2 h and 30 min. The extracted essential oil was dried by anhydrous sodium sulphate and stored in sealed vials at 4°C before antibacterial activity testing, Gas Chromatography-Mass Spectrometric (GC-MS) and Gas Chromatography/Flame Ionization Detector (CG/FID) analysis. The average yield of the extracted essential oil was 1.46±0.05% (w/w). The percentage yield of cinnamon essential oil was calculated using the following formula:

CG/SM and GC/FID analysis: The GC/MS and GC/FID analysis was performed by Sarl Pyrenessences Analysis (Belcaire, France). Briefly, the cinnamon oil was analyzed using a Hewlett Packard 5973, with HP INNOWAX polar column (60 m×0.25 mm×0.25 μm). One microliter essential oil solution diluted in ethanol 10% was injected and analyzed. Helium was the carrier gas with a flow rate of 30 psi/FID; 23 psi/MS. The temperature was programmed at 60°C for 6 min and then increased by 2°C min^–1 up to 250°C for 10 min. The compounds were identified by a combined search of retention time and mass spectra (NKS library, 75,000 spectra). The percentages were calculated from the peak areas given by the GC/ FID, without the use of correction factor.

Antimicrobial study
Microorganisms: The antimicrobial activity of the essential oil of Cinnamomum aromaticum was evaluated against four strains of Gram-negative bacteria (Escherichia coli F5, Escherichia coli F17, Kluyvera spp. and Klebsiella spp.). The microbial strains used in this study were isolated from neonatal calve’s diarrhea.

Preparation of inoculum: Prior to the experiment, bacterial strains were inoculated onto the surface of MacConkey agar media. The inoculum suspensions were obtained by taking five colonies from 24 h cultures. The colonies were suspended in 5 mL of sterile saline (0.85% NaCl) and shaken for 15 sec. The density was adjusted to the turbidity of a 0.5 McFarland Standard (equivalent to 1-5×10⁸ CFU mL^–1).

Antibiotic susceptibility test: Susceptibility to a panel of antimicrobial agents was determined by the standardized disc diffusion assay on Mueller-Hinton agar with commercial antimicrobial susceptibility discs according to the standardization of antimicrobial susceptibility testing in the veterinary medicine at the national level according to WHO recommendations^14,15. The tested antibiotics and their corresponding disc concentrations were as follows: Amoxicillin+acid clavulanic (20/10 μg), ampicillin (10 μg), gentamicin (10 μg), tetracycline (10 μg), colistin (10 μg), trimethoprime/sulfamethoxazole (1.25/23.75 μg), ofloxacine (5 μg) and cifotaxime (30 μg). The prepared plates were incubated at 37°C for 24-48 h. The zone of inhibition was recorded and data were interpreted using the standardization of antimicrobial susceptibility testing in the veterinary medicine at the national level according to WHO recommendations^14,15.

Minimum Inhibitory Concentration (MIC) measurement: Screening of essential oil for antibacterial activity was achieved by the incorporation method as described by Amarti et al.¹⁶, with slight modifications. The essential oil was dissolved in tween 20, which is prepared in sterile distilled water (1/9 v/v). A serial dilution of cinnamon essential oil was prepared with tween 20 (1/10, 1/20, 1/40, 1/80, 1/160 and 1/320). Each test tube contained 4.5 mL of Mueller Hinton agar medium to which were added aseptically 0.5 mL of the prepared dilutions to obtain a final concentrations of 1/100, 1/200, 1/400, 1/800, 1/1600, 1/3200 v/v. The tubes were thoroughly agitated to disperse the essential oil in the culture medium before pouring in to petri plates. The control contained only the culture medium and the tween 20. The plates were inoculated and incubated at 37°C for 24 h. The Minimum Inhibitory Concentration (MIC) was determined by identifying the plates with the lowest concentration of essential oil on which the strain did not grow. Tests were repeated in triplicate. All MIC values are expressed in v/v.

RESULTS

Chemical composition of essential oil: The results concerning the chemical analysis of the essential oil identified by GC-FID were given in Table 1. In general, the composition was relatively homogeneous. Eighty nine compounds were identified, representing 99.99% of total components. E-cinnamaldehyde (94.67%) was the major component followed by coumarin (0.88%) and cinnamyl acetate (0.74%) (Table 1).

Table 1:	Composition of the essential oil of cinnamon

Table 2:	Antibiotic susceptibility of tested strains
R: Resistant, S: Sensitive, I: Intermediate, A: Escherichia coli F17, B: E. coli F5, C: Kluyvera spp., D: Klebsiella spp.

Antibacterial activity
Antibiotic susceptibility: Regarding the antibiotic susceptibility, all tested strains were susceptible to cifotaxime, colistin and gentamicin. However, three strains were resistant to trimethoprime/sulfamethoxazole and two strains were resistant to ofloxacine (Table 2).

Table 3:	Antibacterial activity of essential oil of cinnamon
- : Inhibition, +: Development

Antimicrobial activity of essential oil (determination of MIC): Table 3 showed the results of the antibacterial activity of the essential oil of C. aromaticum. This essential oil exerted strong antibacterial activity. Concentration of 1/1600 v/v was sufficient to inhibit the growth of Escherichia coli F17, Escherichia coli F5, Kluyvera spp. and Klebsiella spp.

DISCUSSION

In the present study, the average yield of cinnamon essential oil was 1.46±0.05% (w/w). A similar result was obtained by Kasim et al.¹⁷ with an average yield of 1.82%. Whereas the levels of the essential oil of Cinnamomum cassia Bark, from Guangxi and Guangdong Provinces (South China), ranged between 1.69±0.67 to 3.21±0.12% (w/w)¹⁸. Similarly, higher yields were recovered by Geng et al.¹⁹ (2.7-3.3% (w/w)), Li et al.²⁰ (2.38%) and Huang et al.²¹ (2.76% (v/w)). Likewise, Jeyaratnam et al.²² obtained also a high yield (2.55% (w/w)) of essential oil from C. cassia by using microwave-assisted hydrodistillation.

Previous studies have shown that the branch bark fraction tended to yield more essential oil compared to the entire branch indicating that selecting the bark based on the tree growth stages as well as separating the stem bark into top, center and lower sections within a tree can significantly improve the extraction efficiency of essential oils²³.

The results of GC/FID were given in Table 1. It can be seen that trans-cinnamaldehyde was the major constituent of cinnamon bark essential oil (94.67%) followed by coumarin (0.88%) and cinnamyle acetate (0.74%). Several studies have mentioned that trans-cinnamaldehyde was the most abundant component in the essential oil C. aromaticum^18,21,24-27, however, the authors recorded different concentrations. For example, the rates of trans-cinnamaldehyde in the essential oil of C. aromaticum analyzed by CG/MS were 97.7, 92.2, 76.9, 72.23, 68.52 and 49.33% as reported by Singh et al.²⁴, Giordani et al.²⁵, Liu et al.²⁶, Poaty et al.²⁷, Deng et al.¹⁸ and Huang et al.²¹, respectively. While Chang et al.²⁸ reported that cis-2-methoxycinnamic acid and cinnamaldehyde were the major compounds of the essential oil of C. aromaticum analyzed by CG/MS (43.06 and 42.37%, respectively). Adinew²⁹ mentioned that 2-propanol, 3-phenyl was the major constituent of cinnamon bark essential oil (analyzed by GC-MS and FT-IR) growing in South West of Ethiopia.

The chemical components of cinnamon oil have been extensively studied in the literature. Bruneton³⁰ found that the main component of the cinnamon (Cinnamomum aromaticum) bark oil was E-cinnamaldehyde and the content was 90%. It is reported that this essential oil contained little eugenol. Similar results were found in this study (94.67% of E-cinnamaldéhyde and 0.01% of eugenol).

Different extraction processes and assay methods could have contributed to differences in cinnamaldehyde levels of cinnamon essential oils³¹. Moreover, the geographical position, cultivation, variety of cinnamon, harvesting time and extraction method affect the effective yield and composition of the essential oil^29,32. Medicinal plants have long been, over the years, played a key role in the human health and contributed to the development of modern therapeutic drugs^33,34. In addition, plant essential oils and extracts have been used for many thousands of years in food preservation, pharmaceuticals, alternative medicine and natural therapies. Therefore, it is necessary to investigate scientifically these plants used in traditional medicine.

Essential oils are potential sources of novel antimicrobial compounds especially against bacterial pathogens²⁵. In this study, the essential oil of cinnamon showed a strong activity against selected bacterial strains. This study showed a MIC at 0.625 μL mL^–1 against 4 strains causing neonatal diarrhea in young calves. Lu et al.³⁵ reported similar results, with a MIC ranging between 0.1-0.4 μL mL^–1. Several studies showed that the essential oil of cinnamon exhibited strong and consistent inhibitory effects against various pathogens by Matan et al.³⁶ and Prabuseenivasan et al.³⁷.

Huang et al.²¹ reported higher values of MIC of essential oils of C. cassia against two Gram-negative bacteria, namely S. typhimurium and E. coli, with concentration of 10 mg mL^–1.

Moreover, Mith et al.³⁸ showed a MIC of essential oils of C. cassia with 0.5/1 μL mL^–1 and 0.25/0.25 μL mL^–1 against E. coli O157:H7 ATCC 35150 and E. coli O157:H7 S0575, respectively.

Chao et al.³⁹ tested the antimicrobial activity of 45 essential oils on bacteria, fungi and yeast and found that cinnamon bark oil exhibited a high and broad-spectrum antimicrobial property. This strong antibacterial property of the essential oil of cinnamon is mainly due to its constituents, including cinnamaldehyde, which particularly displayed strong antibacterial activity compared to the other components^24,40. In addition, Unlu et al.⁴¹ reported that cinnamaldehyde is a main component of the essential oils of cinnamon, this component was effective against some Gram positive and Gram-negative bacteria. Furthermore, Nazzaro et al.⁴² reported that cinnamaldehyde has at least three mechanisms of action against bacteria. At low concentrations, it inhibits enzymes involved in cytokine interactions or other less important cell functions and at higher concentrations, it acts as an ATPase inhibitor. At a lethal concentration, cinnamaldehyde perturbs the membrane. Some studies have reported conflicting information’s on the membrane-perturbing activity of cinnamaldheyde. For example, a sub-lethal concentration of the molecule does not affect the integrity of the membrane in E. coli but can inhibit the growth and bioluminescence of the microorganism Photobacterium leiognathi; this suggests that cinnamaldehyde gains access to the periplasm and perhaps also to the cytoplasm. Cinnamaldehyde is indeed capable of altering the lipid profile of the microbial cell membrane.

CONCLUSION

The present study revealed that Cinnamon aromaticum essential oil is potentially good source of antibacterial agents and could be an alternative for the treatment of diarrhea in calves caused by the tested strains. Further in vivo investigations and clinical trials are needed to justify the potential use of this oil as an antibacterial agent against bacteria causing neonatal calve’s diarrhea.

ACKNOWLEDGMENTS

The authors would like to express their special thanks to the staff members Laboratory of Research on Local Animal Products, Ibn-Khaldoun University of Tiaret (Algeria). The authors are also grateful to Dr. Hamri Mokhtar and Dr. Rezki Hamza for their collaboration during this study.