B. Kaskatepe*, M.E. Kiymaci, D.Simsek, H. B. Erol and S.A. Erdem1

Department of Pharmaceutical Microbiology, Turkey
11Department of Pharmacognosy, Ankara University Faculty of Pharmacy, Ankara, Turkey

*Corresponding Author:
B. Kaskatepe
Department of Pharmaceutical Microbiology, Turkey
E-mail:bkaskatepe@ankara.edu.tr
Date of Submission 24 May 2016
Date of Revision 09 July 2016
Date of Acceptance 20 July 2016
Indian J Pharm Sci 2016;78(4):541-548  

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DOI: 10.4172/pharmaceutical-sciences.1000150

Abstract

There has been an increased interest in essential oils during recent years in accordance with new treatments against pathogens. The aim of the present study was to investigate the contents and compare the antimicrobial activity of different brands of commercial oils with two natural cinnamon oils obtained from the barks. Antibacterial and antifungal activity of cinnamon oils were estimated using disc diffusion and macro dilution methods against Enterococcus faecalis ATCC 19433, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922, Escherichia coli ATCC 35218, Staphylococcus aureus ATCC 29213, Staphylococcus aureus ATCC 25923, methicillin resistant Staphylococcus aureus ATCC 43300, Pseudomonas aeruginosa ATCC 9027, Pseudomonas aeruginosa ATCC 27853, Bacillus subtilis ATCC 6633, Klebsiella pneumoniae RSKK 574, Candida albicans ATCC 10231, Candida albicans ATCC 033. The essential oils compositions were illuminated by GS-MS. Trans-cinnamaldehyde was the major compound of the essential oils obtained from the barks (C4 and C5; 92.3 and 90.1% respectively). The results of the commercial oils have revealed that these oils can be accepted as artificial oils. All the oils were showed antimicrobial activity in a range of doses thought to be from cinnamic aldehyde content.

Keywords

Antimicrobial activity, cinnamon, essential oils

Essential oils have been used in medical, cosmetic and food industries for a long time. In recent years there has been an extensive research to explore and determine the antimicrobial activity of essential oils. Certain studies tilt to natural antimicrobial substances in recent decades because of the resistance to antibiotics. Besides, consumer preference for natural products or products including fewer chemicals in health and food sector has directed the scientific attention towards that area. Lots of essential oils were found to be effective against both standard and clinical bacterial strains. Researchers show that when combined with an antibiotic, essential oils increase their effect besides being alone [1-3]. However, little quantitative data is available on the antimicrobial activity of commercial essential oils and antimicrobial effects are seldom mentioned in their product advertisements. Dussault et al. indicated that shelf life may have effect on the antimicrobial potential of essential oils [4]. But, not enough study has been done in this regard. Moreover purity grades are not specified on commercial packages of many brands.

Cinnamon oil is one of the most studied essential oils because of its high affectivity and redolence. The genus Cinnamomum which belongs to Lauraceae family is represented by approximately 250 species. Among these species C. verum (syn. C. zeylanicum, true cinnamon) and C. cassia (Chinese cinnamon, cassia) are known and mostly used as a spice. Both of these species are also used to provide essential oils. Cinnamon is known as spice since 4000 BC from Chinese writings [5] and also used in herbal medicine for treatment of some diseases like respiratory problems and diabetes. Various biologically active compounds have been identified in cinnamon such as cinnamaldehyde, cinnamic acid, cinnamyl acetate, eugenol and others. Due to their characteristic and biological properties, these chemical compounds have some antimicrobial, antiulcer, antidiabetic, antiinflammatory and antioxidant properties. Cinnamaldehyde is one of the main components of cinnamon and an aromatic organic compound that gives specific taste and odor [6-8]. It is also less toxic (toxic for dividing cells at high concentrations), fat soluble, permeable across living cell membranes, easily undergoes degradation and accepted as biocompatible [9].

Cinnamaldehyde is suggested as a potential candidate for chemoprevention against Helicobacter pylori related gastric pathogenic problems [10]. It is possible to use this compound in several areas like regenerative medicine and tissue engineering in low concentrations, and in neoplastic growth inhibition based on its various medicinal effects at high concentrations [9]. Cinnamaldehyde also affects the structure of the yeast cells by inhibiting mycelial growth and has fungicidal activities by membrane damage against Candida albicans [11]. Moreover, it was reported to have dose-dependent anti-Candida albicans effect. In some concentrations, cinnamaldehyde was found more effective and in some other concentrations similar to fluconazole and nystatine [12]. Besides, trans-cinnamaldehyde shows the ability to inhibit uropathogenic Escherichia coli (UPEC) biofilm formation of UPEC on catheters. Some studies observed that cinnamaldehyde damages membrane integrity and permeability of Staphylococcus aureus (ATCC 3101) and E. coli (ATCC 8735) [13].

Being known as “Generally Recognized as Safe” (GRAS) compounds, nowadays some components of essential oils have been thought as postharvest preservatives against microorganisms[14]. Composition, structure, as well as functional groups of the compounds found in the oils play an important role in determining their antimicrobial activity[15]. Antunes et al. indicated that commercial essential oils have lower biological activity due to the yield process[16]. But there are no studies to verify or contradict these findings worldwide. For this reason, with this study, we aimed to investigate antimicrobial activity of cinnamon oils against to the most common human infectious bacteria. Besides the composition of the selected oils was determined by gas chromatography (GC) and gas chromatographymass spectrometry (GC/MS) analysis.

Two cinnamon bark and three cinnamon oil samples belonging to different commercial brands were purchased from a local market in Turkey. Cinnamon barks were coded as C IV and C V. Essential oils of the barks were obtained by hydrodistillation using Clevenger type apparatus.

Analyses of the cinnamon oils were done simultaneously by GC and GC/MS. GC analysis was performed on an Agilent 6890N Network GC system and GC/MS analysis was performed on Agilent 5973 Network Mass Selective Detector integrated with the GC system. The analysis was performed using HP-Innowax column (60.0 m×0.25 mm×0.25 mm) and helium as carrier gas (1.2 ml/min). The oven temperature was set to 60° for 10 min after injection, then increased to 220° with 4°/min heating ramp for 10 min and increased to 240° with 1°/min heating ramp without hold. Both injector and detector (FID) temperatures were 250°; split ratio was adjusted to 50:1. Injection volume was 2.0 μl. MS conditions were as follows: ionization energy, 70 eV; ion source temperature, 280°; interface temperature, 250°; mass range, 34-450 atomic mass units.

Identification of the components was done by comparison of their relative retention indices and mass spectra with corresponding data[17] and by comparison of their mass spectra with Wiley and NIST Standard Reference Database. The percentages of the components were calculated from the GC peak areas, using the normalization method.

Microorganisms that were selected for this study are Enterococcus faecalis ATCC 19433, E. faecalis ATCC 29212, E. coli ATCC 25922, E. coli ATCC 35218, S. aureus ATCC 29213, S. aureus ATCC 25923, methicillin resistant S. aureus ATCC 43300, Pseudomonas aeruginosa ATCC 9027, P. aeruginosa ATCC 27853, Bacillus subtilis ATCC 6633, Klebsiella pneumonia RSKK 574, C. albicans ATCC 10231, C. albicans ATCC 033.

The bacteria were grown in Mueller Hinton Broth (MHB, Merck) and Mueller-Hinton Agar (MHA, Merck) media. Sabouraud Dextrose Agar (SDA, Merck) was used as growing medium for C. albicans.

Agar disc diffusion and broth dilution method were performed for determination of antimicrobial activity of oils. For agar disc diffusion method, microorganisms were cultured at 37° for 24 h and bacterial and fungal suspensions were prepared from these fresh cultures and adjusted the turbidity equivalent to McFarland 0.5 standard with nephelometer (Biosan). Microorganism suspension was spread on the MHA plate with sterile swab. Sterile discs (6 mm diameter) were impregnated with 15 μl of the essential oils and placed on the surface of the plate. Plates were subsequently incubated at the appropriate temperature for 16-20 h and zones of inhibition were calculated by measuring the diameter in mm.

Minimum inhibitory concentrations (MIC) of oils were determined by the broth macrodilution method. For this purpose two-fold dilutions of essential oils were obtained in tubes including MHB and fresh bacterial and fungal suspension were added. Before analysis, essential oils were homogenized in dimethyl sulfoxide (DMSO). The tubes were incubated 16-20 h at 37°. The lowest concentration that inhibits growth was determined as MIC value. All the experiments were carried out in duplicate and the results were given as mean value. The results were analyzed statistically by SPSS packet program with one way ANOVA test. At 95% confidence interval, P< 0.05 was considered statistically significant.

The composition of the obtained and commercial cinnamon oils which were identified by GC/MS are given in Table 1. Essential oils obtained from the cinnamon barks by hydrodistillation with the yield of 1.5% for C4 and 2.0% for C5. Thirteen and twenty-five compounds were detected representing 97.4% (C4) and 98.2% (C5) of the essential oils obtained from cinnamon barks. Both of the oils were dominated by aromatic hydrocarbons 95.2% for C4 and 89.3% for C5. Other than this, C4 was found to contain oxygen containing monoterpenes (1.9%) and monoterpene hydrocarbons (0.3%), while C5 was found to contain oxygen containing monoterpenes (3.9%) and sesquiterpene hydrocarbons (0.9%) and hydrocarbons (4.3%). The main component was trans-cinnamaldehyde for C4 and C5 with the amount of 92.3% and 86.5%, respectively. C4 and C5 have 1,8-cineole (0.4 and 0.3%, respectively), benzaldehyde (0.7 and 0.2%, respectively), bornyl acetate (0.3 and 0.9%, respectively), terpinen-4-ol (0.3 and 0.7%, respectively), α-terpineol (0.7 and 1.0%, respectively), hydroxyl cinnamic aldehyde (1.0 and 0.7%, respectively), cis-cinnamaldehyde (0.9 and 0.7%, respectively), cinnamyl acetate (2.5 and 0.8%, respectively), carvacrol (both 0.2%), and coumarine (0.3 and 0.4%, respectively) in common. As remarkable difference; monoterpene hydrocarbon content of C4 (α-pinene and limonene) and sesquiterpene hydrocarbon (α-copaene, β-caryophyllene, α-humulene, α-muurolene, δ-cadinenecalamenene and α-calacorene) and hexadecanoic acid with a notable amount (4.1%) for C5 can be observed.

Compounds RIa Cinnamon I Cinnamon
II		 Cinnamon
III		 Cinnamon
IV		 Cinnamon
V
α-pinene 992 - - - 0.1 -
Limonene 1148 - - - 0.2 -
1,8-cineole 1154 - - - 0.4 0.3
Cyclohexanone 1226 - - - - 0.1
α-copaene 1476 - - - - 0.1
Benzaldehyde 1481 1.0 - 0.2 0.7 0.2
Linalool 1487 - 1.5 - - 0.1
Bornyl acetate 1546 - - - 0.3 1.0
Terpinen-4-ol 1558 - - - 0.3 0.7
β-caryophyllene 1583 - - - - 0.3
α-terpineol 1647 - - - 0.7 1.0
Borneol 1677 - - - - 0.4
α-muurolene 1703 - - - - 0.1
δ-cadinene 1733 - - - - 0.2
Hydroxycinnamic aldehyde 1738 - - - 1.0 0.8
2,4-decadienal 1763 - - - - 0.1
Anethole 1780 - - - - 0.2
Calamenene 1801 - - - - 0.2
Benzyl alcohol 1820 - 4.0 -   -
cis-cinnamaldehyde 1854 - - - 0.9 0.7
α-calacorene 1887 - - - - trb
trans-cinnamaldehyde 2007 76.9 10.3 99.5 92.3 90.1
Triacetin 2100 22.1 - -   -
Cinnamylacetate 2099 - - - 2.5 0.8
Eugenol 2111 - - - - 0.1
Carvacrol 2147 - - - 0.2 0.2
Cinnamaldehyde propylene glycol acetalisomer 2210 - 39.0 -   -
Cinnamaldehyde propylene glycol acetal isomer 2226 - 31.0 -   -
Cinnamyl alcohol 2241 - - - - 0.1
Coumarine 2423 - - - 0.3 0.4
Monoterpenehydrocarbons - - - 0.3 -
Oxygen containing monoterpenes - 1.5 - 1,9 4.0
Sesquiterpene hydrocarbons - - - - 0.9
Oxygen containing sesquiterpenes - - - - -
Aromatic hydrocarbons 77.9 84.3 99.7 95.2 93.1
Hydrocarbons 22.1 - -   0.2
Total identified 100 85.8 99.7 97.4 98.2

Table 1: Percentage composition of the Essential Oils obtained from Cinnamon Barks and Commercial Oils

Li et al.[18] reported essential oil analyses of 6 different C. cassia and a C. verum samples; the yield of the essential oils were ranged from 0.72 to 3.08%, due to the variations in the climates, ecological environments and growth conditions in the different cultivation sites; these results are similar with our yields. Transcinnamaldehyde was found between 66.28-77.21% for C. cassia whereas it was found 74.49% for C. verum. Eugenol amount was very low for C. cassia samples (tr- 0.21%), but C. verum was found to contain in noticeable amount (7.29%). In another study, GC/MS analysis of essential oils from the barks of C. zeylanicum (C. verum) were resulted with an amount of 97.7% transcinnamaldehyde together with monoterpenes such as α-pinene, limonene, 1,8-cineole and sesquiterpenes α-copaene, α-amorphene and δ-cadinene [6].

In literature, some studies determined that cinnamon has remarkable antimicrobial activity against some skin infection agents, and food pathogens [4,19-21]. Moreira et al. found that essential oil of C. zeylanicum has a synergistic activity with b-pinene and suggested that these compounds may be an alternative to be inserted in pharmaceutical products as antimold agents [22]. Ooi et al. found that both oil and pure cinnamaldehyde of C. cassia were equally effective in inhibiting the growth of bacteria including Gram positive and Gram negative and fungi. They concluded that broadspectrum antibiotic activities of C. cassia oils are due to cinnamaldehyde [23]. Siddiqua et al. found that cinnamaldehyde and clove oils showed effective antibacterial activity and when combined, the activity increased [24]. Yossa et al. reported that cinnamaldehyde was highly effective against E. coli O157:H7 and Salmonella [25].

Commercial cinnamon oils were previously discussed [26]. As a brief evaluation, cinnamon 2 was found to contain cinnamaldehyde propylene glycol acetal which is a synthetic compound used as a flavor especially in chewing gums[27]. Cinnamon 3 consists of cinnamic aldehyde (99.5%) and cinnamon 1 contains triacetin (22.1%) addition to cinnamic aldehyde (76.9%). Triacetin is not a natural component of essential oils; it is used as diluent or carrier of flavors [28]. Commercial oils were found to contain cinnamaldehyde as major compound, the other compounds were limited with benzaldehyde, benzyl alcohol or linalool, whereas composition of the oils obtained from the barks consist of cinnamaldehyde as major compound like the commercial oils, additionally they are found to contain monoterpenes or sesquiterpenes. Thus, commercial oils can be accepted as artificial oil.

In this work, commercial cinnamon oil 1 and 3, natural cinnamon oil 4 and 5 showed effective antimicrobial activity. Our GC/MS study revealed that cinnamaldehyde was the predominant active compound found in commercial and natural cinnamon oils. Similar to this study, other studies showed that the antibacterial activity of cinnamon oil was due to cinnamaldehyde which was found as major component [29].

Three brands of cinnamon oil had antibacterial and antifungal activity. C2 was the less active essential oil against tested microorganisms. Two of three commercial oils and two natural cinnamon oils were the most active. The antimicrobial activity results of the commercial and natural cinnamon oils are given as zone diameter and MIC values in Table 2.

Microorganisms I II III IV V
ZD MICa ZD MICa ZD MICa ZD MICa ZD MICa
E. faecalisATCC 19433 20 0.78 17 12.5 33 1.56 28 0.39 32 <0.09
E. faecalisATCC 29212 30 0.39 15 12.5 34 0.78 32 <0.09 35 <0.09
E. coliATCC 25922 38 0.09 20 12.5 39 0.09 33 <0.09 25 0,39
E. coliATCC 35218 33 0.19 14 12.5 36 0.09 26 <0.09 35 0,39
S. aureusATCC 29213 37 0.19 13 6.25 40 0.19 34 <0.09 37 <0.09
S. aureusATCC 25923 50 <0.09 15 6.25 45 0.09 35 0.19 42 0.19
S. aureusATCC 43300 50 <0.19 24 6.25 >50 <0.09 42 <0.09 41 <0.09
P. aeruginosaATCC 9027 20 0.78 15 12.5 22 0.78 20 1.56 22 0.78
P. aeruginosaATCC 27853 18 1.56 18 12.5 21 0.78 21 1.56 24 0.78
B. subtilisATCC 6633 40 0.09 19 6.25 40 <0.09 31 <0.09 36 <0.09
K. pneumoniaeRSKK 574 35 0.09 19 6.25 39 <0.09 34 <0.09 43 <0.09
C. albicansATCC 10231 >50 <0.09 >50 3.12 >50 <0.09 >50 <0.09 >50 <0.09
C. albicansATCC 033 >50 <0.09 >50 1.56 >50 <0.09 >50 <0.09 >50 <0.09

Table 2: Inhibition Zone diameter and mic values of Cinnamon Oil

In our study, we investigated the antimicrobial activity of different commercial brands of oils. The most effective of all the three was cinnamon oil 3 (C3). Maximum zone diameter for this oil was observed on C. albicans strains and methicillin resistant S. aureus ATCC 43300 (>50), minimum was on the P. aeruginosa ATCC 27853 (21 mm). Second most effective essential oil was cinnamon oil 1 (C1). It showed maximum zone diameter on C. albicans strains (>50 mm), MRSA 43300, S. aureus ATCC 25923 (50 mm) and minimum zone diameter on P. aeruginosa ATCC 27853 (18 mm). When compared these two oils in terms of content, both of the oils contains cinnamic aldehyde as major component while benzaldehyde was the common minor component. The antimicrobial effects of these oils are thought to be from cinnamaldehyde. The results are consistent with the studies in the literature [18]. C2 comes in the third place in terms of activity level and it is quite different from the other two. Referring to a content analysis, cinnamaldehyde propylene glycol acetal which is a synthetic resin component especially used as a flavoring agent in chewing gums was observed. This oil is believed to be artificial oil. Containing the lower amount of cinnamic acid (10.3%) than C1 and C3 (76.9% and 99.5%, respectively) is believed to be the reason of weaker antimicrobial activity. Also C1 which has lower cinnamaldehyde than C3, C4 and C5 has lower antimicrobial activity. When compared the results of two obtained cinnamon oils, similar results were obtained for both.

When the antimicrobial susceptibility test results were analyzed statistically, there was significant difference between cinnamon oil brands 1 and 2, 2 and 3 (P values were P=0.007 and P=0.003 respectively). There were no statistical differences between C1, C3 and C4, C5.

Also, these commercial available essential oil contents have been found to vary from brand to brand and this situation affects the antimicrobial activity. Being subjected to specific standards and stating contents on the label of these commercial preparations when used as a food additive and cosmetic product by external use, will provide more detailed information in terms of the quality, efficacy and safety of products. This will ensure the more conscious use of these products.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References