Chemical Composition and Antioxidant, Antimicrobial, and Antiproliferative Activities of Cinnamomum zeylanicum Bark Essential Oil

This study examines the chemical constituents, antioxidant potential, antibacterial mechanism, and antiproliferative activity of Cinnamomum zeylanicum bark essential oil. The compositions of the oil were analyzed by GC-MS, and the major constituents were found to be (E)-cinnamaldehyde (71.50%), linalool (7.00%), β-caryophyllene (6.40%), eucalyptol (5.40%), and eugenol (4.60%). C. zeylanicum essential oil contained remarkable levels of phenolic and bioactive compounds with outstanding ability to scavenge free radicals and inhibit β-carotene oxidation. The growth of pathogenic and spoilage bacteria, especially Gram-positive ones (i.e. Listeria innocua, Staphylococcus aureus, and Bacillus cereus), was highly inhibited by the oil, compared to the Gram-negative pairs (i.e. Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhi). The cells of L. innocua and E. coli (as the most sensitive and resistant strains to the oil, respectively) treated with C. zeylanicum essential oil were observed by scanning electron microscopy to unravel structural changes. It was observed that the essential oil quickly exerted its antibacterial activity through disrupting cell envelope and facilitating the leakage of intracellular compounds. The essential oil had also a dose-dependent antiproliferative effect on adipose-derived mesenchymal stem cells (AT-MSCs), and the cell proliferation could be induced by low concentrations of the oil. The present study indicated that C. zeylanicum essential oil with remarkable antioxidant and antimicrobial properties could be applied to develop novel natural preservatives for food and medicinal purposes.


Introduction
Bacteria resistant to antibiotics-derived infections are nowadays considered as one of the largest problems faced by medicine and food industry, due to the requirement for more difficult, cumbersome, and costly processes to diagnose and treat the related severe infections [1].
Currently, many studies have focused on the evaluation of natural products as the source of novel biologically active compounds, owing to the deficiency of common antimicrobial agents to treat infectious diseases [2,3]. In the last two decades, there has been an increasing attention in the use of medicinal herbs to develop new herbal medicines or nutraceuticals. e herbal medicines are rich in bioactive molecules (e.g., polyphenols, carotenoids, and flavonoids) with therapeutic effects, such as delaying the onset of some diseases like cardiovascular disorders, diabetes, and cancer [4].
Essential oils derived from medicinal herbs have versatile applications in ethnomedicine, cosmetics, food, beverages, preservation, fragrance, and pharmaceutical industries. ese bioactive compounds indicate several positive biological properties, such as antioxidant, antiviral, antibacterial, antifungal, insecticidal, and anticancer activities [5].
Cinnamomum zeylanicum belongs to the Lauraceae family that grows wild in India, Sri Lanka, Indochina, and Madagascar. Its inner bark has been used as a potent therapeutic agent in ethnomedicine and as a flavoring ingredient in foods [6]. e antioxidant, antimutagenic, and antimicrobial activities of C. zeylanicum extracts have been previously evaluated [7,8]. However, to our knowledge and as seen in the literature, there are little published reports unraveling the mechanism behind the antibacterial activity of C. zeylanicum bark essential oil against the most resistant and sensitive food-borne pathogenic and spoilage bacteria to the oil. erefore, the present study aimed to assess the antibacterial activity of C. zeylanicum essential oil through a mechanistic approach to provide more information about its antibacterial mechanism. e in vitro antiproliferative effect of the essential oil on adipose-derived mesenchymal stem cells (AT-MSCs) was also investigated.

Essential
Oil Isolation. C. zeylanicum bark (Mashhad, Iran) was subjected to hydrodistillation extraction technique to isolate its essential oil. Dried bark (20 g) was transferred to a Clevenger-type apparatus, and the extraction process was run for 3 h. e resultant essential oil was collected in clean glass vials, dried by anhydrous sodium sulfate, and stored at 4°C until analyses [6].

Gas Chromatography-Mass Spectrometry (GC-MS).
e chemical compounds of C. zeylanicum essential oil were identified by a GC coupled with a mass spectrometer (TRACE MS, ermo Finnigan) with a DB-5 capillary column (30 m × 0.25 mm, 0.25 μm stationary phase thickness). e column temperature was increased from 40°C to 250°C with the heating rate of 2.5°C·min −1 . Helium was applied as the carrier gas, and the ionization energy was 70 eV. e retention indices and mass spectra of the constituents of the essential oil were compared with the published data in the literature, followed by calculation of the retention indices using a homologous series of n-alkane (C 8 -C 20 ) indices [9,10].
e functional groups of the essential oil constituents were analyzed by a FTIR spectrometer. e essential oil was mixed with potassium bromide, and the obtained mixture was compressed to form an appropriate pellet.
e FTIR spectrum of the oil was obtained at 500-4000 cm −1 with 4 cm −1 scanning resolution.

Total Phenolic Contents (TPCs).
e TPC of C. zeylanicum essential oil was determined by means of Folin-Ciocalteu reagent based on a method described by Abeysekera et al. [11]. 20 μL of the essential oil was added to 110 μL of freshly prepared Folin-Ciocalteu reagent (10-time diluted) which was followed by adding sodium carbonate solution (70 μL), incubating at ambient temperature for 30 min, and recording its absorbance at 765 nm. Gallic acid (0.06-1 mg·mL −1 ) was used to formulate the standard curve, and the TPC of the essential oil was then expressed as mg gallic acid equivalent (GAE)/g dry weight C. zeylanicum bark.

DPPH Radical Scavenging
Activity. An aliquot of 37.5 μL of the essential oil or methanol (control sample) was charged and mixed with 2 mL of the DPPH methanolic solution (10 −4 M). e samples were incubated in the dark for 20 min followed by measuring their absorbance at 517 nm against methanol. e following equation was used to calculate the DPPH radical scavenging activity of the essential oil [12]: DPPH radical scavenging activity (%) � 1 − sample absorbance control absorbance × 100. (1)

β-Carotene Bleaching
Assay. e procedure of Ribeiro-Santos et al. [10] was used to perform this assay. 1 mL of β-carotene solution (2 mg β-carotene in chloroform) was mixed with Tween 40 (200 mg) and linoleic acid (20 mg). e chloroform was evaporated by vacuum drying of the mixture at 40°C and then oxygenated ultrapure water (50 mL) was added. Afterwards, the solution was vigorously shaken to form a linoleic acid/β-carotene emulsion, which (5 mL) was subsequently incorporated with the essential oil (200 μL). e resultant mixture was heated in water bath for 120 min, and its absorbance was then recorded at 470 nm (A S120 ). e control sample was prepared according to the same procedure, but methanol was used instead of the essential oil. e absorbance of the control sample was also

Disc Diffusion Agar
Method. e microbial suspension was streaked onto the Petri dishes containing MHA. e essential oil (20 μL) was added to soak the filter paper discs with 6 mm in diameter, and the discs were then placed on the inoculated plates, followed by incubation at 37°C for 24 h. e inhibition zone diameter around filter paper discs was measured in millimeters [6].

Well Diffusion Agar Method.
In this assay, the microbial suspension was spread onto MHA medium in Petri dishes using a L-shaped spreader. Afterwards, several wells (6 mm in diameter) were created on the medium surface and loaded with the essential oil (20 μL). e Petri dishes were kept at a constant temperature of 37°C for 24 h, and the diameter of the inhibition zones around the wells was measured and expressed in millimeters [13].

Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC).
MIC is defined as the lowest essential oil concentration that results in no visible (no turbidity) bacterial growth, and MBC is defined as the lowest essential oil concentration with initial inoculum bacteria killed (no colony formation) [14]. e method of Behbahani et al. [15] with minor changes was applied to measure the MIC and MBC of C. zeylanicum essential oil.
Firstly, the sequential concentrations of the essential oil (100, 50, 25, 12.5, 6.25, 3.125, 1.562, 0.78, and 0.39 mg·mL −1 ) were prepared in MHB medium and sterilized using 0.45 μm syringe filters. Each concentration (200 μL) of the oil was added to the wells (in 96-well plates) which were previously filled with 20 μL of microbial suspensions. After incubation of the plate at 37°C for 24 h, triphenyltetrazolium chloride solution (5%, 20 μL) was added and the plate was reincubated. e lowest concentration of the oil with microbial growth-suppression effect, indicated by the lack of dark-red color in the wells, was considered as the MIC.
One hundred microlitre of the solution in the wells with no microbial growth was then cultured onto the plates containing MHA medium. e plates were subsequently stored at 37°C for 24 h, and the lowest concentration of the oil that killed the bacterial strains, confirmed by the lack of visible colonies on the medium surface, was regarded as the MBC.
2.6. Antimicrobial Mechanism of Action. According to the MIC results, L. innocua and E. coli were selected as the most sensitive and resistant bacteria to the essential oil, respectively, in order to investigate the antimicrobial mechanism action of the oil on their cell membranes.
e bacterial strains were cultured in a broth medium containing MIC of the oil for each bacteria, while the medium being shaken at 37°C. e microbial suspension was centrifuged (5000 ×g for 5 min) to separate the bacterial strains, followed by washing the strains twice by 0.1 M sodium phosphate buffer (pH 7) and filtration using polycarbonate filters. Glutaraldehyde solution (2.5% v/v) was then used to fix the filtrate, and the solution was incubated at 4°C for 120 min. Double distilled water was applied to wash the sample, and ethanol (at increasing concentrations of 30%, 50%, 70%, 80%, 90%, and 100%) was successively used to dehydrate the sample for 10 min. Afterwards, the sample was vacuum dried to evaporate ethanol, coated with gold, and analyzed by means of a scanning electron microscopy apparatus (LEO 1450 VP model, Germany) to check the morphology of the bacterial strains upon treatment with the essential oil [16].

Cell Viability Assay.
Antiproliferative effect of the essential oil against AT-MSCs was analyzed in 96-well flatbottom plates using MTT [3-(4,5-dimethylthiazolyl)-2,5diphenyl-tetrazolium bromide] assay, utilizing the method of Yousefi et al. [17] with some modification. AT-MSCs from healthy donors were obtained and (1 × 10 5 per well) were seeded in the plates until 50-60% confluence was obtained, followed by replacing the medium with 2 mL of complete culture medium (Dulbecco's Modified Eagle's Medium-+ fetal bovine serum) and adding different concentrations of the oil (1, 3.125, 6.25, 12.5, 25, 50, 100, and 200 mg·mL −1 ) into each well. e plates were incubated for 24 h, and cell proliferation was then quantified by MTT assay. Each well was loaded with 30 μL of 5 mg·mL −1 MTT solution, and the plates were incubated in a CO 2 incubator for 3 h. e medium was discarded gently, and the wells were charged with 200 μL of dimethyl sulfoxide. e absorbance of the mixture was finally read at 570 nm using an ELISA reader (Convergent Technologies, Marburg, Germany).

Statistical
Analysis. All data were processed by one-way analysis of variance (ANOVA) in SPSS software. e results were reported as mean ± standard deviation, and the experiments were replicated three times.

Total Phenolic Content.
Phenolic compounds, as a major group of phytochemicals, have great importance owing to their antioxidant activity. e essential oil C. zeylanicum had a TPC of 106.19 ± 0.63 mg GAE/g dried essential oil. A TPC of 0.91 [23], 48.90 [24], 53.74 [25], and 912 mg GAE/g [26] was reported for C. zeylanicum essential oil. ese controversial results suggest that the composition and quality of essential oils from plant sources are strongly influenced by the age and variety of the plant, geographical conditions, drying methods, and the extraction procedures used to isolate the essential oils [27].

Antioxidant Activity.
e antioxidant activity of essential oils is most likely attributed to a synergy among their constituents, and the major constituents are primarily responsible for this positive biological effect of essential oils [28]. In this work, DPPH-RS activity and β-carotene bleaching assays were used to determine the antioxidant activity of C. zeylanicum essential oil. e DPPH-RS activity assay is based on the reaction of potent antioxidants with DPPH stable free radicals with intense violet color and subsequently converts them to a colorless compound; thus, the discoloration degree indicates the free radical scavenging activity of the antioxidant agent [29]. e DPPH-RS activity of the essential oil of C. zeylanicum was found to be 71.12 ± 0.77%, indicating the strong ability of the oil to neutralize DPPH free radicals via either hydrogen atom or electron donation mechanisms [30]. e β-carotene bleaching assay was employed to evaluate the potential of C. zeylanicum essential oil to prevent lipid peroxidation. It is a useful and practical assay due to the fact that it is performed in an emulsion and many food products tend to be emulsified during their productions [31]. e C. zeylanicum essential oil had a high inhibitory effect (63.08 ± 0.81%) against β-carotene discoloration. is means that the oil is able to scavenge and neutralize linoleate free radicals that are responsible for β-carotene discoloration [32]. e present results are in line with the findings of other studies [10,11], and the antioxidant activity of the essential oil is mainly ascribed to its phenolic and other bioactive compounds. For example, the antioxidant activity of (E)-cinnamaldehyde [33], α-pinene [34], eugenol [35,36], β-caryophyllene [37], and eucalyptol [38] has been reported in the literature. It is therefore possible to deduce that the C. zeylanicum essential oil had strong radical scavenging characteristics along with the potential to suppress lipid oxidation reaction. e oil could be used as a natural alternative to the synthetic antioxidants in the food preservation technologies in order to improve the oxidative stability of many food products. e antibacterial activity of C. zeylanicum essential oil was investigated towards some pathogenic Gram-positive (i.e. L. innocua, S. aureus, and B. cereus) and Gram-negative (i.e. S. typhi, P. aeruginosa, and E. coli) bacterial species, using disc diffusion agar, well diffusion agar, MIC, and MBC methods. e oil indicated a strong antibacterial effect towards all the microorganisms tested, as indicated in Table 2. It had a bacterial type-dependent antimicrobial activity; the highest and lowest inhibition zones were observed for L. innocua and E. coli, respectively, according to the results of disc/well diffusion agar tests. Moreover, a greater antibacterial activity or inhibition zone was found in the well diffusion agar compared to the disc diffusion agar assay. Indeed, the bacterial species in the well diffusion agar method are in direct contact to the essential oil, but the diffusion rate of the antimicrobial agent from the disc surfaces to the medium determines its inhibitory effect in the disc diffusion agar test [9,13]. e MIC and MBC values of C. zeylanicum essential oil against the aforementioned bacteria are also reported in Table 2. As can be clearly observed, the Gram-positive bacteria were growth-suppressed or killed in the presence of lower concentrations of the essential oil compared to the Gramnegative ones, mainly due to the presence of a single mucopeptide layer in their cell membranes that makes them to be more sensitive to the antimicrobial agents. In contrast, the cell membranes of Gram-negative bacteria contained a more complexed lipopolysaccharide and phospholipid layer with remarkably lower diffusion rate to lipophilic-based antimicrobial compounds of the essential oils [1,4].
Similar observations have been previously reported by researchers [6,39]. e compound cinnamaldehyde, as the main chemical constituent of C. zeylanicum essential oil, has been implicated in the antimicrobial effect towards Gramnegative/positive bacteria and fungi species, through inhibiting the formation of essential bacterial enzymes and/or causing intense damage to the bacterial cell walls [40,41]. Hence, the high antibacterial potential of C. zeylanicum essential oil could be attributed to its high cinnamaldehyde concentration. Figure 2shows the microbial cells before and after treatment with C. zeylanicum essential oil. e cell appearances were changed upon treatment, likely through altering outer cell envelopes. e untreated E. coli cells have their typical striated wall structures (Figure 2(a)), and the differences between the control cells and the treated ones can be easily observed in the rod morphology (Figure 2(b)). It seems that the essential oil of C. zeylanicum has the ability to disrupt the membrane of E. coli cells and facilitate intracellular compounds leakage, as indicated by the presence of malformed and sunken cell shapes. In fact, the essential oil resulted in a high cell disruption and subsequently greater cell lysis degrees in E. coli. e strained wall structures of L. innocua cells were changed after treatment with the essential oil, as well (Figures 2(c) and 2(d)). e oil caused deformity in the cell surfaces, and the treated L. innocua cells had wrinkled and irregular cellular shapes. e present results have confirmed the detrimental effect of C. zeylanicum-originated essential oils against the cell wall of E. coli and L. innocua.

Antibacterial Mechanism of Action of the Essential Oil.
e corresponding changes in cell walls of bacterial species could be due to the membrane lysis and transformations induced by the damage on the membrane integrity and permeability from the essential oil [14]. It seems that C. zeylanicum essential oil could act on the membrane and lead to a marked change in its lipid profile and increase in its surface areas, thereby altering its structure. It also has the potential to penetrate the deeper part of the cells and facilitate their death rate [40].

Antiproliferative Effect.
e antiproliferative activity of C. zeylanicum essential oil was investigated against AT-MSCs using MTT assay. MSCs, as multipotent self-renewing cells, have the potential to differentiate towards various cell/ tissue lineages and produce growth-improving secretomes with antioxidant and anti-inflammatory agents, thereby making them to be used as an appropriate candidate in regenerative medicine [17]. e cells were exposed to increasing doses of the essential oil ranging from 1 to 200 mg·mL −1 for 24 h. e essential oil had a concentration-dependent growth-inhibition effect on AT-MSCs, and the antiproliferative activity of the oil was increased markedly as its concentration increased up to 200 mg·mL −1 (Figure 3). e IC 50 value for the antiproliferative effect of the oil on AT-MSCs was calculated to be 83.51 mg·mL −1 . It seems that low concentrations of C. zeylanicum essential oil are able to induce cell proliferation and are beneficial for AT-MSC growth, besides having an outstanding antioxidant activity and strong bactericidal effect (at MBC < 50 mg·mL −1 ) against all pathogenic and spoilage bacteria tested in this study.
Essential oils could exert their antiproliferation through several mechanisms including (i) disrupting cell membrane integrity via depolarization, permeability increment, or reducing membrane-bound enzymes activity, (ii) altering mevalonate metabolism pathway, or (iii) inducing apoptosis [5]. e antiproliferative effect of C. zeylanicum essential oil has been previously shown against different cell lines, such as

Conclusion
e results of the present research showed that the essential oil obtained from C. zeylanicum is rich in bioactive compounds, and its main chemical constituent is (E)-cinnamaldehyde, comprising 71.5% of the total oil composition. e essential oil conferred superb antioxidant activity and antibacterial effect, especially towards Gram-positive bacteria. A dose-dependent antiproliferative activity was also observed when AT-MSCs were exposed to the essential oil at dosages from 1 to 200 mg·mL −1 for 24 h. Results have shown to be very positive and indicated good potential of the oil for use in food products as a natural bioactive ingredient. However, further studies are required to unravel the  Evidence-Based Complementary and Alternative Medicine mechanisms underlying the antiproliferative activity of the essential oil of C. zeylanicum and to scale up its application in many food products.

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

Conflicts of Interest
e authors declare no conflicts of interest.