Abstract: The aim of this study is to determine the antimicrobial activity of Cinnamomum verum stem bark aqueous extract against food-borne pathogen bacteria, nosocomial infection bacteria and normal flora. Extraction with an aqueous system from the dried stem barks of C. verum yielded 2.5% of the dried plant. Among 10 test strains of bacteria, C. verum showed inhibitory effect on the growth of Krebsilla pneumoniae ATCC 10031, Straphylococcus epidermidis ATCC 12228 and E.coli ATCC 25922 in an agar diffusion test. The Minimal Inhibitory Concentrations (MICs) and the Minimal Bactericidal Concentrations (MBCs) were in the range of 4-16 and 16-32 g L-1, respectively. In conclusion, C. verum stem bark aqueous extract showed interesting inhibitory effect on the growth of S. epidermidis, K. pneumoniae and E. coli at low minimum concentration. This may give additional information of antimicrobial activity of C. verum stem bark aqueous extract.
INTRODUCTION
The food-borne pathogen is the major concern in food production factories especially for ecporting countries such as Thailand and Vietnam. There are reports on the food borne- pathogen bacteria in food products in Thailand (Padungtod et al., 2008) andan increase of antibiotic resistance in food-borne pathogen bacteria in Vietnam (Tillotson et al., 2008; Gerner-Smidt and Whichard, 2008; Dowzicky and Park, 2008; Van et al., 2007). This is the reason why food preservative compound is added to food products.
In nature, plants contain a variety of compounds called phytochemical and some of them have medicinal properties. Long time ago humanity learned to used plants for disease treatment or control. Today, scientific research reveals that not only the chemical from the plant has effect against a particular disease, but, that the antioxidant property of the plant’s extract also gives beneficial effect to human health.
The use of food preservatives today is strictly regulated because of toxicity. In addition, chemicals as food preservative (indicated by labeling in food packages) is a major concern for consumer (Mau et al., 2001). Therefore, natural antimicrobial compounds from plant have to be explored. Since, cinnamon is a common food ingredient and hence probably non-toxic, it is very interesting to evaluate the biological activity of this plant.
Cinnamomum verum (Syn Cinnamomum zeylanicum Blume), or cinnamon belongs to the family Laureceae. Cinnamon is a wooden tree that grows in tropical Asia and Africa. The bark of the tree has a special smell andis usually used as a spice in food and dessert recipes. In western countries, the extract of cinnamon bark is used as an aroma powder. Cinnamon is rich in essential oils and tannins which inhibit microbial growth (Mau et al., 2001). Cinnamon essential oil has been reported to have antimicrobial activity against Escherichia coli o157:H7 (Senhaji et al., 2007), Listeria monocytogenes, Salmonella choleraesuis, Aspegillus flavus, Candida albican (Lopez et al., 2007). In addition, the mixture of clove and cinnamon essential oil showed antimicrobial activity against food born pathogen bacteria (Aspergillus flavus, Pennicillium roqueforti, Mucor plumbeus, Eurotium sp., Debaryomyces hansenii, Pichia membranaefaciens, Zygosaccharomyces rouxii and Candida lipolytica) in modidified condition (Matan et al., 2006). The essential oil part of cinnamon showed significant inhibitory effect on the growth of food-borne pathogen bacteria. However, the antibacterial activity of the aqueous extract of this plant has never been reported. The objective of the present study is to investigate the antimicrobial activity of C. verum aqueous extract against various bacteria.
MATERIALS AND METHODS
Tested Bacteria
The micro-organisms used in this study consist of five gram positive (Staphylococcus
aureus ATCC 25923, Staphylococcus epidermidis ATCC 12228, Micrococcus
luteus ATCC 9341, Bacillus subtillis ATCC 6633, Lactobacillus
plantarum ATCC 14917) and five gram negative (Escherichia coli ATCC25922,
Salmonella typhimurium ATCC 14028 Klebsiella pneumoniae ATCC 10031
Proteus vulgaris ATCC 13315, Pseudomonas aeruginosa ATCC 9721
). All are reference strains obtained from the American Type Culture Collection.
Among the bacteria, there are food-borne pathogen bacteria (S. aureus ATCC 25923, E. coli ATCC 25922 and S. typhimurium ATCC 14028), nosocomial infection bacteria (K. pneumoniae ATCC 10031, P. vulgaris ATCC 13315 and Ps. aeruginosa ATCC 9721) and normal flora bacteria (L. plantarum ATCC 14917 and S. epidermidis ATCC 12228) that were included in the susceptibility test.
Plant Material
The dried stem barks of Cinnamomum verum were bought in April 2008
from a local Chinese medicine shop in the Mahasarakham province, Thailand. Identification
was confirmed by the Department of Biology, Faculty of Science, Mahasarakham
University, Mahasarakham, Thailand.
Extraction
Ten grams of dried stem bark of C. verum were boiled in 1 L of water.
For each dried stembark the extraction was repeated 3 times. The filtrate of
the extraction was spray dried into powder form. The yield of extraction was
2.5% of the dried plant.
Antimicrobial Assay
Agar Diffusion Susceptibility Test
The antimicrobial activity of C. verum was evaluated using the agar
diffusion method as described in the standard guideline (Lorian,
1996). The spray dried powder of plant extract was prepared into solutions
by dissolving in sterile water at concentrations of 125, 250 and 500 mg mL-1.
Three hundred microliter of plant solution were injected into sterile stainless
steel cylinders (6 mm internal diameter and 10 mm height) that were placed on
the inoculated Mueller Hinton agar (MHA) surface. After pre-diffusion at room
temperature for 1 h, the plates were incubated at 37°C for 19 h. The Normal
Saline Solution (0.9% NaCl) (NSS) injected into the cylinder was used as control
and a 10 μg mL-1 gentamicin sulphate (Sigma Chemical Co., St.Louis,
USA) solution was used as standard in the same cultivation plate. The inhibition
zones were measured and data are mean of three measurements.
Mics and MBCs Determination Using Agar Dilution and Broth Macro Dilution
Methods
MICs for C. verum aqueous extract was determined by the agar dilution
method (Lorian, 1996) while MBCs were determined by the
broth macro-dilution method (Lorian, 1996) andusing gentamicin
sulphate as reference antibiotics (Sigma Chemical Co., St. Louis, USA). Briefly,
inoculates were prepared in the same medium at density adjusted to 0.5 McFarland
turbidity standard (108 colony-forming units (cfu) mL-1)
and two-fold dilution for the broth macro-dilution procedure. The inoculated
tubes were incubated at 37°C and the MICs were recorded after 24 h of incubation.
The MIC was defined as the lowest concentration of plant extract or gentamicin
sulphate at which the microorganism tested did not showed visible growth, while
MBC was defined as the minimum bactericidal concentration with negative subcultures
on the agar medium.
RESULTS AND DISCUSSION
The result in Table 1 shows that the plant extract inhibited the growth of Krebsilla pneumoniae ATCC 10031, Straphylococcus epidermidis ATCC 12228 and E. coli ATCC 25922. The Minimal Inhibitory Concentrations (MICs) were in the range of 4-16 mg mL-1 while Minimal Bactericidal Concentrations (MBCs) were in the range of 16-32 mg mL-1, respectively (Table 2).
Food-borne pathogen bacteria cause problems for food industry and consumers. Food-borne disease can be the cause of serious health problems and even death in humans (Busani, 2006). The food borne pathogen bacteria was found to contaminate live stock production countries such as Thailand (Padungtod et al., 2008) and Vietnam (Van et al., 2007). Bacteria that often contaminate food are S. aureus, K. pneumoniae, S. typhymurium, Campylobacter sp. and Listeria sp. (Padungtod et al., 2008).
In earlier study, the antimicrobial activity of C. verum essential oil against food-borne pathogen bacteria was reported and was found that the essential oil of C. verum showed inhibitory effect against Staphylococcus aureus, Enterococcus faecalis, Listeria monocytogenes, Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa, Yersinia enterocolitica, Salmonella choleraesuis, Aspergillus flavus, Penicillium islandicum and Candida albicans (Lopez et al., 2007; Senhaji et al., 2007; Becerril et al., 2007; Matan et al., 2006; Mau et al., 2001; Singh et al., 2007).
| Table 1: | Inhibition zone diameters of C. verum aqueous extract against various bacteria |
| Data are mean±SD (n = 3); nz: No inhibition zone | |
| Table 2: | The MICs and MBCs of C. verum aqueous extract against various bacteria |
| nd: Not determined | |
Additionally, in vivo antitumorigenic activity of C. verum has been reported (Amara et al., 2008). The major chemical constituents in C. verum reported are thymol, carvacol, carvone, cinnamaldehyde, eugenol and linalool as components in its essential oil (Friedman et al., 2000). However, the antimicrobial activity of C. verum aqueous extract has never been reported. The results of the present study indicate that C. verum showed inhibitory effect against Krebsilla pneumoniae ATCC 10031, Straphylococcus epidermidis ATCC 12228 and E. coli ATCC 25922. This finding also indicates that the C. verum aqueous extract showed better inhibitory effect on the growth of gram negative bacteria than gram positive bacteria. Due to the difference of cell wall composition between gram positive and gram negative bacteria, this may indicate that the C. verum aqueous antimicrobial activity should involve the cell wall of the bacterium because it showed inhibitory effect in gram negative bacteria more than gram positive bacteria. The result gives additional data for the antimicrobial activity of C.verum aqueous extract. As mention before, this is the first reported of antimicrobial activity of C. verum aqueous extract. In conclusion, C. verum aqueous extract showed antimicrobial activity against Krebsilla pneumoniae ATCC 10031, Straphylococcus epidermidis ATCC 12228 and E. coli ATCC 25922 at low concentration. This might give additional data to support the use of C. verum against food-borne pathogen bacteria.
ACKNOWLEDGMENT
This study received partial financial support from the Faculty of Science, Mahasarakham University, Thailand.