Agricultural Sciences in China

2011, 10(9): 1482-1487

September 2011

Antibacterial Effect of Cinnamon Oil Combined with Thyme or Clove Oil

LU Fei1, DING Yi-cheng1, YE Xing-qian2 and DING Yu-ting1
1 2

College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou 310014, P.R.China Department of Food Science and Nutrition, Zhejiang University, Hangzhou 310029, P.R.China

Abstract
Antibacterial effect of cinnamon oil combined with thyme or clove oil was studied in this paper. Agar dilution method was used to determine the minimum inhibitory concentrations (MICs) of the essential oils (EOs) of cinnamon, thyme, and clove oil against three Gram-positive bacteria (Bacillus subtilis, Bacillus cereus, and Staphylococcus aureus), and two Gramnegative bacteria (Escherichia coli and Salmonella typhimurium). The results showed that cinnamon was a promising antibacterial substance with MIC ranged from 0.1 to 0.4 L mL-1 for the five bacterial species. Agar dilution checkerboard method was used to test the combined antibacterial effect of cinnamon oil with thyme or clove oil. Combination of cinnamon and thyme oil showed an additive effect against all selected bacteria, and combination of cinnamon and clove oil displayed an additive effect against B. subtilis, B. cereus, S. aureus, and an indifferent effect against E. coli and S. typhimurium. Furthermore, gas chromatography-mass spectrometry (GC-MS) measurement was used to analyze the components of the EOs, and the main components of cinnamon, thyme, and clove were cinnamaldehyde, thymol, carvacrol and p-cymene, and eugenol, respectively. Key words: cinnamon oil, antibacterial effect, essential oils, combination, chemical components

INTRODUCTION
Cinnamon (Cinnamomum zeylanicum or Cinnamomum verum), rich in essential oils (EO), belongs to Lauraceae family and usually grows in South and South-East Asia. C. zeylanicum oil has strong antimicrobial activity amongst essential oils of plants from Lauraceae family (Raharivelomanana et al. 1989; Mishra et al. 2008). Chao et al. (2000) 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. The antimicrobial activity of EOs of cinnamon, clove, basil, rosemary, dill, and ginger on four Gram-positive bacteria (Staphylococcus aureus, Bacillus cereus, Enterococcus

faecalis, and Listeria monocytogenes), four Gram-negative bacteria (Escherichia coli, Yersinia enterocolitica, Salmonella choleraesuis , a n d P s e u d o m o n a s aeruginosa), and three fungi (a yeast, Candida albicans, and two molds, Penicillium islandicum and Aspergillus flavus) was evaluated, and the results showed that cinnamon and clove gave the strongest inhibition (Lopez et al. 2005). Furthermore, Muthuswamy et al. (2008) found that 10 L mL-1 cinnamon bark ethanolic extract solution could inhibit the growth of E. coli O157:H7 and L. innocua in apple slices. Combined utilization of antimicrobials has the potential of an increased antimicrobial effect, a side effect reduction and a decreased microbial resistance to antimicrobials. Synergistic effects of combined utilization of different EOs were recently reported con-

Received 14 October, 2010

Accepted 13 May, 2011

Correspondence DING Yu-ting, Professor, Tel/Fax: +86-571-88320237, E-mail: dingyt@mail.hz.zj.cn

2011, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S1671-2927(11)60142-9

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firming their improved antimicrobial effect without rising their concentrations (Bidlack et al. 2000; Gutierrez et al. 2008). Combined extracts prepared from cinnamon (Cinnamomum cassia), Chinese chive (Allium tuberosum), and corni fructus (Cornus officinalis) exhibited a better inhibition on growth of E. coli than potassium sorbate (Mau et al. 2001). The antibacterial effect of cinnamon has been largely studied, and it has been proved to be a strong antimicrobial. However, few studies have been published on the antibacterial effect of combined application of cinnamon oil with other EOs. The present study was aimed to find out the antibacterial effect of cinnamon in combination with thyme and clove oil. Firstly, the chemical components of these EOs were analyzed. Secondly, the antibacterial effect of cinnamon, thyme, and clove oil was studied using the agar dilution method and then the antibacterial effect of the combination of cinnamon with other EOs was investigated.

min-l to 250C for 5 min was used for the analysis. The chemical components were identified by comparing their mass spectra with National Institute of Standards and Technology, USA, mass spectra literature and several authentic references. The compounds were quantified by integrating the peak area of the spectrograms. Each EO was tested in triplicate, and values are presented as meanstandard deviation of replicated measurements.

Bacteria
Three Gram-positive bacteria of Bacillus subtilis (ATCC6633), Bacillus cereus (ATCC10876), and Staphylococcus aureus (ATCC6538), and two Gram-negative bacteria of Escherichia coli (ATCC11229) and Salmonella typhimurium (ATCC14028) were offered by Zhejiang Provincial Center for Disease Control and Prevention (CDC) (Hangzhou, China). The strains were cultured twice in nutrient broth (NB) at 37C for 24 h, then streaked on nutrient agar slants and cultured under the same conditions. The slants were stored at 4C and subcultured monthly until use. The strains were adjusted to the required concentration of 108 CFU (colony forming units) mL-1 by using the McFarland standard.

MATERIALS AND METHODS

Cinnamon oil and other EOs
Cinnamon, thyme, and clove oil were supplied by Flowers & Herbs Limited Company (Shanghai, China), and all of them were extracted by steam distillation method (Table 1).

Assessment of the minimum inhibitory concentration of EOs by means of agar dilution method
The values of minimum inhibitory concentration (MIC) of the EOs were determined by the agar dilution method (ADM) (Yoshida et al. 1997). MIC was defined as the lowest concentration that did not result in any visible growth of the microorganism in comparison with the growth in the control plate; the presence of one or two colonies was not taken into account for final assessment of MIC (Hammer et al. 1999). The results of the antibacterial effect of the EOs against the five bacterial strains expressed as MIC (L mL-1). Values are given as mean of tests performed in triplicate.

Chemical components analysis of EOs

2 mL of essential oils was used for gas chromatography-mass spectrometry (GC-MS) measurement. GCMS analysis was performed on an Agilent6890N gas chromatographer with an HP 5973 mass spectrometer detector. HP-5Ms capillary column (30.0 m0.25 mm0.25 m) was used. Helium (99.999%) was the carrier gas with a flow rate of 1.0 mL min-1, the injection volume was 1 L, and split ratio was 1:1. A temperature program of 100C for 3 min followed by 20C
Table 1 Essential oils selected in this study
Essential oils Cinnamon Thyme Clove Scientific name Cinnamomum zeylanicum Thymus vulgaris Eugenia caryophyllata

Part of plant Bark of cinnamon Flower and leaf of thyme Bud of clove

Extraction method Steam distillation Steam distillation Steam distillation

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Assessment of the combined antibacterial effect of cinnamon oil by means of agar dilution checkerboard method
Serial two-fold dilutions of cinnamon (0.025-0.4 L mL -1 ) and thyme (0.05-0.8 L mL -1) or cinnamon (0.025-0.4 L mL-1) and clove (0.05-0.8 L mL-1) were mixed (White et al. 1996) according to the same ADM adopted to assess MIC. Subsequently, 10 L of the nutrient broth containing 108 CFU mL-1 of the indicator strains were inoculated to all plates, and each plate was inoculated 6 times evenly. Plates were then incubated at 37C for 24 h. The analysis of the combination of the substances was obtained by calculating FICI as follows: FICI=FICa (MICa of the combination/MICa alone)+FICb (MICb of the combination/MICb alone), where a is cinnamon oil and b is thyme or clove oil in the combination. The FICI is interpreted as: (1) a synergistic effect when FICI 0.5; (2) an additive effect when FICI>0.5 and 1; (3) an indifferent effect when FICI>1 and 4; (4) an antagonistic effect when FICI>4 (Gutierrez et al. 2008). All measurements were performed in duplicate and then replicated, and the mean values are given.

RESULTS AND DISCUSSION

Chemical components analysis of EOs
Tables 2, 3, and 4 show the chemical components of cinnamon, thyme, and clove oil, respectively, identified by GC-MS. Twelve components were identified
Table 2 The analysis of the chemical components of cinnamon oil
No. 1 2 3 4 5 6 7 8 9 10 11 12 Components Benzaldehyde Benzeneacetaldehyde 2-Methoxyphenol Formic acid, phenylmethyl ester 3-Phenyl-, 2-propenal cis-Cinnamaldehyde trans-Cinnamaldehyde Caryophyllene -Caryophyllene 1-Naphthalenol, 2-methylBenzyl cinnamate Cinnmyl cinnamate Total content

in cinnamon oil. The main components of cinnamon oil were trans-cinnamaldehyde, cinnmyl cinnamate, and benzyl cinnamate of which the relative contents were 77.348, 13.999, and 1.377%, respectively. p-cymene, -terpinene, borneol, and thymol were the main components of thyme, and the relative contents were 20.399, 12.669, 15.882, and 19.173%, respectively, and there were nineteen components identified. There were nine components identified in clove oil, and the main components were eugenol and -caryophyllene, the relative contents were 80.345 and 14.778%, respectively. The chemical components of cinnamon, thyme and clove oil have been studied in literatures. Marongiu et al. (2007) found that the main component of the cinnamon (Cinnamomum zeylanicum) bark oil was transcinnamaldehyde, and the content was 77.1%. The main components of thyme oil were different in different studies. Hazzit et al. (2009) found that there were similar major components of Thymus species (Thymus algeriensis, Thymus pallescens, and Thymus dratensis) which were carvacrol (44.4-57.7%), p-cymene (10.317.3%), and -terpinene (10.8-14.2%). Furthermore, they found that even if the same species, different samples may have different components. Arraiza et al. (2009) and zcan and Chalchat (2004) found that the main components of thyme (Thymus vulgaris L.) were thymol (36.3-47.5%), p-cymene (9.9-27.8%), and terpinene (5.3-16.2%). In our study, the content of thymol was relatively lower than that in the studies of Arraiza et al. (2009) and zcan and Chalchat (2004). The relative content of carvacrol was 4.791% in our study. It has been confirmed that the main compo-

Retention time (min) 4.43 6.09 6.92 7.17 7.27 7.33 7.71 8.36 8.49 8.88 9.02 9.54

Relative content (%) 0.7480.021 0.9270.024 0.8940.032 0.4880.036 0.4800.049 0.8840.051 77.3480.053 0.390.041 0.3190.037 0.780.046 1.3770.014 13.9990.019 98.6340.283

Values represent as meanstandard deviation (n=3). The same as below.

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Table 3 The analysis of the chemical components of thyme oil

No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Components -Pinene Camphene -Pinene Myrcene p-Cymene 1-Octen-3-ol -Terpinene Sabinene hydrate Terpinolene Linalool Camphor Borneol Thymol methyl ether Thymol Carvacrol -Caryophyllene Bisabolene Sesqui-phellandrene Cadinene Total content Retention time (min) 4.07 4.58 4.63 5.01 5.11 5.16 5.24 5.46 5.78 5.84 6.75 6.80 6.91 7.66 7.74 8.86 8.99 9.33 9.45 Relative content (%) 2.3460.102 0.5630.038 0.5770.037 4.0330.052 20.3990.083 2.1680.062 12.6690.201 5.0430.083 3.1210.043 3.2020.021 0.3680.016 15.8820.219 2.5290.086 19.1730.108 4.7910.078 0.6190.039 0.6210.026 0.4810.039 0.4820.048 99.0700.186

nents of clove are eugenol and -caryophyllene as reported by Alma et al. (2007) and Guan et al. (2007).

Combined antibacterial effect of cinnamon oil with thyme or clove oil

As shown in Table 5, cinnamon oil showed the strongest antibacterial effect against B. subtilis, B. cereus, S. aureus, E. coli, and S. typhimurium with the MIC of 0.2, 0.1, 0.1, 0.4, and 0.4 L mL-1, respectively. Thyme and clove oil had the same MIC against E. coli and S. typhimurium (0.8 L mL-1). But, the antibacterial effect of thyme oil was higher than that of clove oil against B. subtilis, B. cereus, and S. aureus. The combined antibacterial effect of cinnamon oil was evaluated by the agar dilution checkerboard method when it was combined with thyme or clove oil against B. subtilis, B. cereus, S. aureus, E . coli , and S. typhimurium. The results are reported in Table 5. The combined application of cinnamon oil with thyme oil displayed an additive effect against B. subtilis, B. cereus, S. aureus, E. coli and S. typhimurium with the FICI of 0.75, 1, 0.75, 0.75, and 0.75, respectively. Additive effect was observed when cinnamon oil was combined with clove oil against B. subtilis, B. cereus and S. aureus, and the combination showed an indiffer-

Table 4 The analysis of the chemical components of clove oil

No. 1 2 3 4 5 6 7 8 9 Components -Cubebene Eugenol -Cadinene -Caryophyllene -Caryophyllene -Cadinene -Farnesene -Cadinene Acetyl-eugenol Total content Retention time (min) 7.64 8.26 8.49 8.87 9.12 9.46 9.51 9.96 10.01 Relative content (%) 0.4770.025 80.3450.207 0.4550.036 14.7780.093 2.0080.056 0.3660.029 0.3040.015 0.2650.012 0.9010.057 99.8900.508

Table 5 Fractional inhibitory concentration (FIC) and FIC indices

Bacteria B. subtilis Related indexes MICo MICc FIC FICI MICo MICc FIC FICI MICo MICc FIC FICI MICo MICc FIC FICI MICo MICc FIC FICI Cinnamon-thyme Cinnamon 0.2 0.05 0.25 0.75 (A) 0.1 0.05 0.5 1 (A) 0.1 0.05 0.5 0.75 (A) 0.4 0.1 0.25 0.75 (A) 0.4 0.1 0.25 0.75 (A) Thyme 0.4 0.2 0.5 0.2 0.1 0.5 0.4 0.1 0.25 0.8 0.4 0.5 0.8 0.4 0.5 Cinnamon 0.2 0.05 0.25 0.75 (A) 0.1 0.05 0.5 1 (A) 0.1 0.05 0.5 0.625 (A) 0.4 0.4 1 1.03 (I) 0.4 0.4 1 1.03 (I) Cinnamon-clove Clove 0.8 0.4 0.5 0.4 0.2 0.5 0.8 0.1 0.125 0.8 0.025 0.03 0.8 0.025 0.03

B. cereus

S. aureus

E. coli

S. typhimurium

1)

MICo, MIC of one sample alone; MICc, MIC of one sample of the most effective combination; FIC, MIC of the combination/MIC alone; FICI, sum of FIC of cinnamon oil and FIC of the combined agent with cinnamon oil. Results are interpreted as synergy (S, FICI 0.5), addition (A, 0.5<FICI 1), indifference (I, 1<FICI 4) or antagonism (AN, FICI>4).

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ent effect against E. coli and S. typhimurium. The major composing substances of cinnamon, thyme and clove oil are cinnamaldehyde, carvacrol and thymol, and eugenol, respectively. The additive effects of the main components have been reported. For example, a mixture of cinnamaldehyde and eugenol at 250 and 500 g mL -1 inhibited growth of Staphylococcus sp., whereas the substrates applied individually did not inhibit growth (Moleyar and Narasimham 1992). The simultaneous application of carvacrol or thymol (0.3 mmol L - 1 or 45 g mL -1) caused a larger decline in viable counts for the strains of B. cereus than that when the antimicrobials were individually applied (Periago et al. 2001). The antimicrobial effect of cinnamon oil combinations has rarely been studied, especially by using agar dilution checkerboard method. Hsieh et al. (2001) researched the antimicrobial effect of combined extracts of corni fructus (Cornus officinalis Sieb. et Zucc.), cinnamon (Cinnamomum cassia Blume) and Chinese chive (Allium tuberosum Rottler) (8:1:1, v/v/v) against bacteria, yeasts, and moulds, and the combined extracts showed an entire antimicrobial spectrum and outstanding inhibitory effect. Gutierrez et al. (2008) used checkerboard method to quantify the combined antibacterial effect of oregano, marjoram, thyme and basil. All the oregano combinations showed additive effect against B. cereus, and oregano combined with marjoram, thyme or basil also had an additive effect against E. coli and Pseudomonas aeruginosa. The mixtures of marjoram or thyme also displayed additive effects in combination with basil, rosemary or sage against Listeria monocytogenes.

Tech R&D Program of China (863 Program) (SQ2007AA10XK140105), the Zhejiang Province Key Scientific and Technological Project, China (2006C12051), and the Zhejiang Provincial Center for Disease Control and Prevention (CDC) (Hangzhou, China).

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CONCLUSION
In this study, it is confirmed that cinnamon oil is a strong antibacterial agents. Cinnamon oil combinations displayed an additive or indifferent effect against the food bacteria, indicating that cinnamon oil may be in combination with other EOs to increase the antibacterial effect of food preservatives, therefore, to increase the food quality and safety.

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Acknowledgements
This study was partially supported by the National High-

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of Cinnamomum zeylanicum: Chemical characterization and antityrosinase activity. Journal of Agricultural and Food Chemistry, 55, 10022-10027. Mishra A K, Mishra A, Bhargava A, Pandey A K. 2008. Antimicrobial activity of essential oils from the leaves of Cinnamomum spp. National Academic Science Letters, 31, 341-345. Moleyar V, Narasimham P. 1992. Antibacterial activity of essential oil components. International Journal of Food Microbiology, 16, 337-342. Muthuswamy S, Rupasinghe H P V, Stratton G W. 2008. Antimicrobial effect of cinnamon bark extract on Escherichia coli O157:H7, Listeria innocua and fresh-cut apple slices. Journal of Food Safety, 28, 534-549. zcan M, Chalchat J. 2004. Aroma profile of Thymus vulgaris L. growing wild in Turkey. Bulgarian Journal of Plant Physiology, 30, 68-73.

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