|Year : 2020 | Volume
| Issue : 2 | Page : 162-171
Mechanism of action and bioactivities of Cinnamomum zeylanicum essential oil against some pathogenic microbes
Eman W Elgammal1, Abd El Nasser G El Gendy2, Abd El-Basset A Elgamal1
1 Department of Chemistry of Natural and Microbial Products, National Research Centre, Giza, Egypt
2 Department of Medicinal and Aromatic Plants Research, National Research Centre, Giza, Egypt
|Date of Submission||14-Dec-2019|
|Date of Acceptance||28-Feb-2020|
|Date of Web Publication||30-Jun-2020|
PhD Eman W Elgammal
Department of Chemistry of Natural and Microbial Products, National Research Centre, Dokki 12311
Source of Support: None, Conflict of Interest: None
Background and objective The emergence of multiple drug-resistant pathogenic microorganisms leads to the search for new, safe, and effective therapeutic substances. Essential oils (EOs) of the cinnamon species are known for their biological antimicrobial and antioxidant properties. Therefore, in our study, the EO extracted from the bark of wild Cinnamomum zeylanicum grown in the green mountains of Oman was investigated to study both the aspects.
Materials and methods Chemical composition of the EO was analyzed by gas chromatography–mass spectrometry, and then its antimicrobial potential was tested against different Gram-positive, Gram-negative bacteria and fungi. The minimum inhibitory concentration (MIC) was detected and a further in-depth study on the antimicrobial mode of action of the EO at the MIC concentration was studied, in addition to the determination of the antioxidant potential of the studied EO.
Results and conclusion Thirty compounds were identified, and the major constituents were cinnamaldehyde (81.78%), bornyl acetate (5.33%), and cinnamyl acetate (2.82%). The antimicrobial results showed a highly significant activity against all tested microbes. The MIC of our EO ranged from 3.3 μl/ml for Gram-positive bacteria and fungi to 10 μl/ml for Gram-negative ones. The mode of action of EO at MIC concentration showed that it had a strong effect on cell viability, permeability of cell membrane, and leakage of cell constituents such as DNA, RNA, and protein from the cell membrane to outside with the lethal percent reaching 99.99%. Moreover, it was observed that the bactericidal and fungicidal effects act in a dose- and time-dependent manner and they depend on the culture condition. Finally, the EO had a strong antioxidant effect with IC50 equaling 2.3 mg. Cinnamon essential oil has a great potential as a useful agent help in combating resistant microorganisms coupled with healing of wounds according to its antioxidant activity.
Keywords: antimicrobial activities, Cinnamomum zeylanicum, essential oils, minimum inhibitory concentration, mode of action and antioxidant effect
|How to cite this article:|
Elgammal EW, El Gendy AG, Elgamal ABA. Mechanism of action and bioactivities of Cinnamomum zeylanicum essential oil against some pathogenic microbes. Egypt Pharmaceut J 2020;19:162-71
|How to cite this URL:|
Elgammal EW, El Gendy AG, Elgamal ABA. Mechanism of action and bioactivities of Cinnamomum zeylanicum essential oil against some pathogenic microbes. Egypt Pharmaceut J [serial online] 2020 [cited 2020 Aug 12];19:162-71. Available from: http://www.epj.eg.net/text.asp?2020/19/2/162/288661
| Introduction|| |
During the last decades of the twentieth century, there was an increase in the incidence of drug-resistant pathogens to common antibiotics, which necessitated extensive research to find new therapeutic agents from other natural resources such as medicinal plants to treat human infectious diseases . So, many plants were investigated for the production of new natural products. These natural products have the ability to inhibit the growth of pathogenic microorganisms, without harming the host, indicating that they will be good alternatives for synthetic antibiotics .
Cinnamon is a plant, belonging to the Lauraceae family. It has shown many biological applications due to its antiseptic, analgesic, antispasmodic, astringent, aphrodisiac, carminative, parasiticide, insecticidal, and hemostatic properties . Furthermore, it is exhibited to have antimicrobial efficacy in its essential oils (EOS) obtained from the branches and barks  because of the presence of trans-cinnamaldehyde which have antimicrobial activity against a wide range of microbial pathogens, with various spectrums of activity . Moreover, oils and extracts from cinnamon possess a distinct antioxidant activity, which is attributed to the presence of phenolic and polyphenolic substances. These previous effects coupled with antioxidant activity could be helpful in the treatment of microbial infection and promoting wounds to heal .
Since the antimicrobial pattern of the EOs is partly dependent on their mechanism of action, our research discussed this point to measure that effect on cell membrane disruption, which occurs by passing any EO components through the plasma membrane causing the leakage of cell constituents . Furthermore, there are many measures for detecting the mode of action such as observing changes in cell morphology using transmission electron microscopy , measuring ATP synthesis, change of intracellular pH , measuring oxygen consumption , and determination of antimicrobial action kinetics . Detailed information on how these compounds work to achieve their antimicrobial effect is still to be explored.
This study aimed to analyze the chemical composition of the EO obtained from wild Cinnamomum zeylanicum bark collected from the green mountains of Oman and assess its inhibitory effect on the growth and survival of some pathogenic bacteria and fungi, in addition to studying its mode of action with different methods.
| Materials and methods|| |
Collection, extraction, and identification of essential oil
Plant material collection and extraction of essential oil
The bark of C. zeylanicum plant was collected from the green mountain of Oman. The plant samples were identified in the herbarium of the Faculty of Science, Mansoura University, Egypt.
The EO of the plant bark was obtained from the entire plant by hydrodistillation using a modified Clevenger-type apparatus for 3 h according to the method of Guenther . The oily layer obtained was separated and dried with anhydrous sodium sulfate. All the EOs were kept in sealed airtight glass vials covered with an aluminum foil and maintained at 4°C until further analysis.
Gas chromatography–mass spectrometry analysis
The gas chromatography–mass spectrometry (GC-MS) analysis of the EO samples was carried out using GC-MS instrument stands at the Department of Medicinal and Aromatic Plants Research, National Research Center with the following specifications. Instrument: a TRACE GC Ultra Gas Chromatograph (Thermo Scientific Corp., Miami, CA, USA), coupled with a Thermo mass spectrometer detector (ISQ Single Quadrupole Mass Spectrometer; Model ISQ Spectrometer, Thermo Scientific Corp., Miami, CA, USA). The GC-MS system was equipped with a TR-5 MS column (30 m×0.32 mm i.d., 0.25 µm film thickness). Analyses were carried out using helium as carrier gas at a flow rate of 1.3 ml/min and a split ratio of 1 : 10 using the following temperature program: 60°C for 1 min; rising at 4.0°C/min to 240°C and held for 1 min. The injector and detector were held at 200°C. Diluted samples (1 : 10 hexane, v/v) of 1 µl of the mixtures were always injected. Mass spectra were obtained by electron ionization at 70 eV, using a spectral range of m/z 40–450.
Identification of essential oil constituents
Identification of the chemical constituents of the EO was deconvoluted using AMDIS software (www.amdis.net) and identified by its retention indices (relative to n-alkanes C8–C22), mass spectrum matching to authentic standards (when available, Wiley spectral library collection and NSIT library database).
Evaluation of antimicrobial potential and mode of action of essential oil
Antimicrobial tests were studied using Gram-positive bacteria such as Bacillus subtilis ATCC6633, Bacillus cereus ATCC6629 and Staphylococcus aureus ATCC29213, Gram-negative bacteria such as Escherichia coli ATCC25922, Salmonella typhimurium ATCC14028, Klebsiella pneumonia ATCC13883, and Proteus vulgaris (isolate) as well as the fungi Candida albicans ATCC10231 and Aspergillus niger obtained from Chemistry of Natural and Microbial Products, National Research Center. Bacteria and fungi were kept at 4°C on Nutrient Agar slants and Sabouraud dextrose agar slants, respectively.
Bacteria and fungi inocula were obtained from overnight cultures grown on nutrient agar and Sabouraud dextrose agar slants, respectively. The cultures were incubated at 37°C for bacteria and 30°C for fungi. A loopful of cells was transferred into sterile saline solution (NaCl 0.85% w/v) to provide a final concentration of ∼106 count forming units per ml adjusted according to the turbidity of 0.5 McFarland scale tube at 600 nm.
Antimicrobial activity of essential oil
Antimicrobial activities of the EO were carried out by agar well diffusion method using the macrodilution approach . The culture media were prepared for the tested microbes as mentioned above. After sterilization, these media were poured in 9 cm Petri dishes containing 100 μl of different inocula prepared as mentioned above. After solidification of the media, 10 mm wells were made and loaded with 100 μl of crude EO and diluted EO which ranged from 5 to 50 μl/ml. Then the inoculated plates were incubated for 24 h at 37°C for bacteria and for 2 days at 30°C for fungi. After the incubation time, antimicrobial activities were detected by measuring the inhibition zone diameter expressed in millimeters (mm) against ciprofloxacin and ketoconazole (0.5 mg/ml) as standards for antibacterial and antifungal activities, respectively. Negative controls were prepared using DMSO used for diluting the EO.
Minimal inhibitory concentration of essential oil
Minimum inhibitory concentrations (MICs) were assessed by serial broth dilution method . The inocula were freshly prepared as mentioned above. The EO was first dissolved in DMSO and then different concentrations were prepared by diluting the samples in the respective media. The concentrations ranges were from 3 to 30 μl/ml. Each one ml from the above concentrations received 0.1 ml of the inoculum to reach 106 CFU/ml for our tested microbes. Triplicate tests were performed, and the average was taken as the final reading comparing negative and positive controls.
Kinetic study and mode of action of essential oil
Viability of microbial cells: the antimicrobial effect of EO on the viability of some pathogens was evaluated applying two different methods as given below.
Kill time analysis at MIC of cinnamon essential oil (CEO) using ordinary culture conditions: B. subtilis ATCC6633, E. coli ATCC25922, and C. albicans ATCC10231 were selected as examples for Gram-positive, Gram-negative bacteria, and fungi, respectively. EO at MIC concentration was added to the cultures (105 CFU/ml) in nutrient broth medium for bacteria and Sabouraud medium for fungi to reach a final volume of 5 ml. Cultures in broth with DMSO only was considered as positive control. The cultures were incubated at 37°C for 6 h at 150 rpm, then samples were taken every 2 h, serially diluted in saline, spread on the surface of the respective media, and then incubated at 37°C for 24 h. Subsequently, the colonies were counted in terms of CFU/ml .
Kill time analysis using different concentrations of CEO: kill-kinetics study using the previous organisms was performed to analyze the kill time of CEO using different concentrations of MIC. Cinnamon oil was diluted into tubes with phosphate buffered saline at 1×MI, 2×MIC, and 3×MIC concentrations. Then, the tubes were inoculated with freshly prepared bacterial suspension in order to maintain the initial bacterial concentration of 105CFU/ml and incubated at 37°C. Samples were removed at 0, 0.5, 1, and 2 h of incubation, and diluted serially. Viable counts were determined by plating the diluted aliquots on respective agar plates and incubated for 24 h at respective temperature. As a control, the bacterial suspension in sterile phosphate buffer solution with DMSO was also tested .
Integrity of the cell membrane: the cell membrane integrity of the same organisms was determined by quantification of cellular materials released into the extracellular medium, especially DNA, RNA, and protein. EO at the MIC concentration was added to 5 ml of the microbial inoculum (105 CFU/ml) in a sterilized medium (nutrient broth for bacteria and Sabouraud for fungi) and then incubated at 150 rpm, 37°C for 6 h. Then the samples were collected after 2, 4, and 6 h of incubation and centrifuged at 4000 rpm for 10 min, and the supernatant was collected for the determination of leakage of intracellular content by measuring the concentration of DNA, RNA, and protein using NanoDrop . EO without bacteria in the culture media was used as the negative control. The untreated cultures were served as positive controls.
Permeability of the cell membrane: the cell membrane permeability in terms of relative electrical conductivity (EC) was performed . Our tested microbes were cultured in broth media and incubated at 37°C for 10 h, then, the suspensions were centrifuged, and the pellets were washed with 5% glucose until its EC became close to that of 5% glucose. CEO at MIC concentration was added to 5% glucose (L1), and to the isotonic bacterial solution, and incubated at 37°C for 2 h, then the EC was measured every 30 min (L2). The control was killed bacteria in 5% glucose (L0). The permeability of the bacterial membrane was calculated as follows:
Evaluation of antioxidant potential by 2,2’-diphenyl-1-picrylhydrazyl free radical scavenging assay
The antioxidant activity of CEO was measured in terms of hydrogen-donating or radical-scavenging ability with some modifications . The tested EO was diluted in methanol (0.25–5 mg) and a volume of 100 μl of it was put into a test tube containing 900 μl of 2,2’-diphenyl-1-picrylhydrazyl (DPPH) of 0.1 mmol/l in methanol. Shaken vigorously and kept in dark for 30 min at room temperature. The control was prepared as above without the EO and methanol was used for zero adjustment. The decrease in absorbance was measured at 517 nm.
Free radical scavenging activity of the tested EO was calculated according to the following formula:
where A sample is the absorbance of DPPH solution with EO and A control is the absorbance of DPPH solution in methanol without EO. Each assay was carried out in triplicate.
| Results|| |
Chemical constituents of the essential oil
In our research, 30 components in CEO were extracted from the bark and are presented in [Table 1]. The main components are cinnamaldehyde (81.78%), bornyl acetate (5.38%), and cinnamyl acetate (2.82%).
|Table 1 Relative percentage of volatile compounds in cinnamon essential oil bark|
Click here to view
In-vitro antimicrobial potential of CEO was assessed by agar well diffusion method ([Table 2]). The results showed that the plant EO was significantly effective against all tested microbes. The EO fully inhibited the growth of all tested organisms except A. niger which is partially inhibited giving an inhibition zone of 33 mm.
|Table 2 Antimicrobial activity of cinnamon essential oil and standard references (0.5 mg/ml) expressed as inhibition diameter zones in millimeters|
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The data obtained from [Table 3] demonstrate microbial susceptibility in relation to oil concentration. It depends on the growth ability of microorganisms under the effect of different concentrations of EO indicating by growth inhibition zone. The values of oil concentrations ranged between 5 and 50 μl/ml. It was noticed that the sensitivity of microorganisms increased with the increment of oil concentration. Different effective concentrations of CEO against the assayed organisms were found.
Minimum inhibitory concentration
[Table 4] shows the evaluation of MIC of CEO against the pathological strains. The MIC values of the oil ranged from 3 to 30 μl/ml. Gram-positive bacteria and fungi were found to be more sensitive than Gram-negative ones. Also, it is noticed that Gram-positive bacteria and fungi have similar sensitivity toward EOs giving the smallest MIC (3.3 μl/ml) value compared with Gram-negative ones (5–10 μl/ml).
|Table 4 Minimum inhibitory concentration (μl/ml) of cinnamon essential oil against pathological organisms|
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Mechanism of action of cinnamon essential oil
Three organisms (B. subtilis ATCC6633, E. coli ATCC25922, and C. albicans ATCC10231) were selected for a further in-depth study of the antimicrobial mode of action of EO. The mode of action of EO against the investigated microbes was studied by several assays, including time-kill analysis, quantity of biomolecules released, and cell membrane permeability.
Study of kill time assay applying ordinary culture conditions
The relationship between microbial cell viability and kill time analyses of CEO under the effect of ordinary culture conditions was investigated for determining the bactericidal or fungicidal effect of the EO. As shown in [Figure 1], EO at the MIC (10 μl/ml for E. coli and 3.33 μl/ml for B. subtilis and C. albicans) concentration showed an increased reduction of viable cell count along with increasing incubation time for all organisms used compared with the control (without EO). However, the EO had positive results against the three tested microbes during the tested time, but the reduction rate was not significant (0.35–2.1 log) and did not reach the total kill point during our incubation time.
|Figure 1 Time-kill curves in control and treated samples with cinnamon essential oil at minimum inhibitory concentration of (a) Bacillus subtilis ATCC6633, (b) Escherichia coli ATCC25922, and (c) Candida albicans ATCC10231.|
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Study of kill time assay applying different concentrations of cinnamon essential oil
From the previous experiment, it was concluded that the ordinary culture condition was effective and enhanced cell viability; therefore, in the following test to confirm that effect, we neglected this factor and used instead phosphate buffered saline.
Time-kill curves at 1×MIC, 2×MIC, and 3×MIC concentrations of CEO for the same microorganisms were cultured in phosphate buffer saline and incubated at 37°C for 2 h as shown in [Figure 2]. The concentration effect relationship has shown that EO at higher MIC values had bactericidal and fungicidal effects and the reduction value was greater than or equal to 3 log CFU/ml.
|Figure 2 Time-kill curves in control and treated samples with cinnamon essential oil at different concentrations of minimum inhibitory concentrations of (a) Bacillus subtilis ATCC6633, (b) Escherichia coli ATCC25922, and (c) Candida albicans ATCC10231.|
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Effects of cinnamon essential oil on cell release
The cell constituents’ leakage under the effect of CEO at MIC concentration was investigated to give further insight into the mode of action of our EO against the three tested microbial examples. In this experiment, nucleic acid and protein leakage were studied in time-dependent test ([Figure 3]). It was observed that the amounts of released DNA, RNA, and protein of all tested microbes increased with the time. The maximum concentration of leaked DNA, RNA, and protein (after subtracting the negative control) in cell suspensions was (1900, 6000, and 3000 ng/μl), (1080, 2920, and 1090 ng/μl), and (110, 150 and 100 mg/μl), respectively for treated B. subtilis, E. coli, and C. albicans compared with the untreated controls (91, 105, and 200 ng/μl), (82, 95, and 165 ng/μl), and (1.6, 3.2, and 4 mg/μl), respectively after 6 h.
|Figure 3 Quantity of biomolecules released by (a) Bacillus subtilis ATCC6633, (b) Escherichia coli ATCC25922, and (c) Candida albicans ATCC10231 treated with cinnamon essential oil.|
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Effects of cinnamon essential oil on cell permeability
Cell membrane permeability was measured in terms of EC as shown in [Figure 4]. The cells treated with CEO at MIC concentration showed high EC compared with the control indicating the effect of CEO on membrane permeability. The increased EC is considered as a result of leakage of intracellular electrolytes. The maximum relative conductivity of 26.1, 28.2, and 31.0 was reached after 2 h.
|Figure 4 Effect of cinnamon essential oil at minimum inhibitory concentration on cell membrane permeability of Bacillus subtilis ATCC6633, Escherichia coli ATCC25922, and Candida albicans ATCC10231.|
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Evaluation of antioxidant potential
Radical scavenging activity of CEO using DPPH is illustrated in [Figure 5] which showed that the antioxidant potential (25±1.4–82.9±2.1%) is dependent on the concentration with IC50 reaching 2.3 mg.
|Figure 5 Antioxidant activity of cinnamon essential oil using 2,2’-diphenyl-1-picrylhydrazyl.|
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| Discussion|| |
EOs of cinnamon bark have been used for thousands of years, in natural therapy, alternative medicine, and in pharmaceutical applications . So, it is necessary to investigate the role of this plant EO in the treatment of common infectious diseases to overcome the emergence of antibiotic-resistant microorganisms . The composition of CEO is greatly influenced by the method of extraction, species of the plant, plant parts, development stage, plant age, geographic origin, and time of harvest  Our results showed that the main components are cinnamaldehyde (81.78%), bornyl acetate (5.38%), and cinnamyl acetate (2.82%). It has been validated by  that transcinnamaldehyde (86.16%) was the major component of CEO of the bark. Furthermore , observed that the major component of CEO extracted from the leaf was cinnamaldehyde which possessed inhibitory activity against some microorganisms by the inhibition of cell enzyme synthesis, disruption of the cell wall structure resulting in shortage in the cytoplasm, cytoplasmic granulation, cytoplasm acidity, and depletion of intracellular ATP collect . And this is in agreement with our obtained results with CEO which had a significant inhibitory effect on all tested microbes.
About the antimicrobial activity, our tested EO was significantly effective against all tested microbes. These findings are in agreement with the results of ,, who reported that CEO was effective against 10 examined bacteria including S. aureus and E. coli. Furthermore  obtained the same result with some Gram-negative, Gram-positive bacteria, fungi, and yeasts which were completely inhibited by cinnamon bark oil. This is because the main component of CEO is cinnamaldehyde, which has been proven to be effective substance against some Gram-negative and Gram-positive bacteria including Pseudomonas, clostridium, and yeasts like Candida strains ,; moreover, the presence of terpenes in CEO is believed to accumulate in bacterial cell membrane and cause a loss of the membrane integrity, cell lysis, and death .
The microbial susceptibility test showed that Gram-positive bacteria and fungi were more sensitive than Gram-negative ones in low concentrations. In that context, the study by  showed different effective concentrations of CEO against S. aureus. Also  obtained the same results with CEO against the tested microbes.
The MIC test showed that Gram-positive bacteria and fungi were more sensitive than Gram-negative ones. This is in agreement with  who tested the CEO of leaves against E. coli, P. aeruginosa, S. aureus, and S. enterica and found that the EO exhibited marked inhibitory effect toward all assayed bacteria. And the smallest MIC (1.25 μl/ml) was against S. aureus (Gram positive) while the highest one was against P. aeruginosa (Gram negative), while  reported that the oil of C. zeylanicum and C. cassia were more effective against E. coli more than P. aeruginos and S. aureus.
Study of kill time assay applying ordinary culture conditions demonstrated that the reduction rate was not significant (0.35–2.1 log) and did not reach the total kill point during our incubation time; this is may be due to the presence of culture medium which not only can protect the microbial cells from the action of EO but also can activate or decrease the EO activity . Overall, the reduction of microbial growth is concentration dependent or time dependent on the applied EO ,. In this context  studied the effect of C. burmannii’s EO on the growth of E. coli, C. albicans, and S. aureus and found that the growth reduction was increased over the time interval compared with the control. Also  applied 0.05% of oregano EO against meat microbial flora cultured in broth for several days at 37°C and they obtained a 2 log decrease in viable cell count and the total kill point was not achieved. Furthermore  found that the CEO decreased the viable cell count of the tested bacteria (E. coli, Listeria monocytogenes, Pseudomonas erogenous, Staphylococcus aureus, and Enterobacter aerogenes) cultured in nutrient broth from 2 to 5 log after 24 h. of incubation compared with the control using concentrations of 10, 20, and 30 µl/ml.
On the other hand, the concentration effect relationship has demonstrated that EO at higher MIC values had bactericidal and fungicidal effects because the reduction value was greater than or equal to 3 log CFU/ml . The concentration of 3xMIC (30 μl/ml for E. coli and 10 μl/ml for C. albicans, B. subtilus) was found to be the most effective ones after half an hour. of incubation and beyond that time the reduction rate in viable cell count was not significant. The reduction reached about 3, 3.6, and 4.7 log with a lethal percent of 99.87, 99.97, and 99.99% for C. albicans, B. subtilus, and E. coli, respectively, after 2 h. This indicates that it had bactericidal effect  acting in a dose-dependent manner ,. This is in agreement with  who reported that cinnamon bark oil had a bactericidal effect against S. aureus with 3.6 log10 reduction in bacterial cell count at a concentration of 4×MIC after 24 h of incubation time. this suggests that the primary mechanism of action of CEO is cell wall lysis  or weakening of the cell wall leading to irreversible damage to the cytoplasmic membrane due to osmotic pressure , or the EO penetrated the cells causing complete lysis and cellular death .
On the other hand, the results showed that Gram-negative bacteria is more resistant than Gram-positive bacteria and fungi, the reduction in viable cells of E. coli reached 2.2 log at 10 μl/ml, while it reached 1.4 and 2.0 log at a concentration of 10 μl/ml for B subtilis and C. albicans, respectively, after 120 min of incubation. The MIC for E. coli was three times more than that of B. subtilis and C albicans and this confirmed our previous obtained results of MIC test.
Effects of CEO on cell release revealed that the amount of released DNA, RNA, and protein of all tested organisms increased with time . The high amount of the released biomolecules after exposure to CEO indicated that the cytoplasmic membrane was damaged leading to cell lysis and death . That is in agreement with , who measured the concentrations of released protein, DNA, and RNA from three strains of Mycobacterium tuberculosis (IS310, IS53, and H37Rv) and found the protein concentration ranges (5 × 104-107 × 104 ng/μl for untreated strains and 37.9×104–1200.9×104 ng/μl in treated strains) were higher than that of released DNA (70 at MIC −506 ng/μl at MBC) and RNA (20 at MIC −151.6 ng/μl at MBC). From our results, there were some differences between the microorganisms which could be related to the differences in plasma membrane and cell wall structure . Our data indicated that, however, E. coli is more resistant to the EO than B. subtilis and C. albicans but it released nucleic acid and protein more than of the other ones; this may be due to the different action of EO on cell damage. This is in accordance with  who treated E. coli APL 87/1 and B. subtilis APL 87/35 with clove and oregano EOs and found that E .coli was damaged by forming holes on the cell surface, whereas B. subtilis damage was just on the cell surface contributing to the malformation.Furthermore, the cell membrane integrity indicated by cell permeability may affect cell metabolism because of the fact that it is integral with the maintenance of cell energy, metabolic regulation, transport of solutes, and control of turgor pressure; so, any slight change in cell integrity may lead to cell death . This is in agreement with  who observed that an increase in EC positively is proportional to plant extract concentration.
Finally, CEO have good free radical scavenging property  that may play an important role in the prevention of some diseases such as cancer, immune system decline, and heart disease and act as anti-inflammatory agent . Furthermore, the antioxidant activity coupled with antibacterial, antifungal, antiallergic, and anticarcinogenic agents  could be a helpful therapeutic agent in promoting wound healing .
| Conclusion|| |
This study has shown that CEO possesses potential antimicrobial activity against several microbes, and it has a good antioxidant activity. Cinnamaldehyde was found to be the major component of CEO which is considered as a natural antimicrobial agent. Moreover, the mechanism of action of CEO at MIC concentration demonstrated that it has bactericidal and fungicidal effects and it could penetrate the cell wall of the pathogens causing their damage and consequent release of cell constituents (DNA, RNA, and protein).Therefore, it has an adverse effect on microbial pathogens and it may be successfully applied as a promising alternative drug for treatment of infectious diseases and help in wound healing. Furthermore, the effective dose to kill microbes is very small which means CEO can be used in many other applications such as food spoilage, the limiting factor to control water contamination by bacteria or fungi.
The authors acknowledge The National Research Centre, Egypt, for supporting this work Financial support and sponsorship.
Eman W. Elgammal, Abd El-Nasser G. El Gendy, and Abd El-Basset A. Elgamal together proposed and designed the study. Eman Elgammal performed the microbiology part and wrote the manuscript. Abd El-Nasser G. El Gendy and Abd El-Basset A. Elgamal performed the chemistry part and collected the samples.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3], [Table 4]