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Assessment of antimicrobial activity of onion extract (Allium cepa) on streptococcus mutans and streptococcus sanguinis; in vitro study.

Introduction

Dental caries and periodontal diseases are considered to be the most common chronic diseases all over the world. Dental caries is defined as an infectious disease with microbial origin that results in destruction of the calcified tissues of the teeth. One group of the bacteria attributed to dental caries is Streptococcus mutans. All eight S. mutans serotypes are both acidogenic and aciduric and are strongly stimulated by sucrose (Roberson, T., H.O. Heymann, 2006). Pioneering animal model studies, as well as human epidemiologic researches, have strongly implicated Streptococcus mutans as the main etiological agents in human dental caries (Kuramitsu, H., 1993). Streptococcus sanguinis is recognized not only for its association with bacterial endocarditis but also because of its known antagonist role in dental caries. This microorganism competes with Streptococcus mutans for colonization sites on tooth surfaces (Caufield, P.W., 2000). Despite the recent decline in dental caries frequency among children in developed countries, tooth decay is still remained as a major health problem. Nevertheless, many populations are still in risk of developing carious lesions (Bauer, A.W., 1966).

Vancomycin is a narrow spectrum antibiotic which is highly effective against gram positive bacteria. It is commonly used for serious infections caused by penicillinase-resistant Staphylococci (Small, P.M., H.F. Chambers, 1990; Gonzalez, C., 1999). Recently the resistance of microorganisms has increased due to indiscriminate use of antimicrobial drugs commonly used for the treatment of infectious diseases (Cohen, M.L., 2000). This problem has created a renewed interest to search for new other antimicrobial substances from various sources including medicinal plants (Bauer, A.W., 1966).

Allium is the largest and an important representative genus of Alliacae family. Onion (Allium cepa) is a bulbous plant widely cultivated in almost every country. Traditionally it has been used to treat intestinal infections. It has been reported to have antibacterial, antiviral, antiparasitic, antifungal properties. It is also antihypertensive, hypoglycemic, antithrombotic, antihyperlipidemic, anti inflammatory, and antioxidant (8). Allium cepa extracts has been widely studied for its antimicrobial activity. Kim et al. reported its antibacterial activity against Streptococcus mutans, Streptococcus sobrinus, Prevotella intermedia and Porphyromonas gingivalis (Kim, J.H., 1997). Benkeblia et al. investigated antimicrobial activity of different concentrations (50, 100, 200, 300, 500 ml/l) of essential oil extracts of three types of onions (green, yellow and red) and garlic against two bacteria, Staphylococcus aureus, Salmonella enteritidis, and three fungi, Aspergillus niger, Penicillium cyclopium, and Usarium oxysporum. Green onion showed the lowest antimicrobial activity (10). Maidment et al. examined a total of twelve Alliums for antibacterial activity against the tested microorganism. Allium cepa did not show antibacterial activity (Maidment, D., 2001).

Considering the controversies in the antimicrobial activity of Allium cepa as reviewed above, the differences of microorganisms in each study reviewed above, the antibiotic resistance, and lack of enough investigations on onions cultivated in Iran, the purpose of this study was to examine in vitro antibacterial activity of Allium cepa extract against Streptococcus mutans, and Streptococcus sanguinis.

Materials and methods

This experimental study was conducted in Shiraz University of Medical Sciences. Three types of onion (green, red and yellow) were purchased from a local market in Shiraz. Vancomycin (Jaber ebne hayyan pharmaceutical company, Iran) was bought from a local pharmacy. Lyophilized ampoule of Streptococcus mutans (ATCC: 35668) and Streptococcus sanguinis (ATCC 10556) was purchased from the microbial collection of Persian Type Culture Collection (PTCC), Iran.

Preparation of onion extract:

1 kg of each type of onion was randomly selected, was washed carefully, and was blended (Panasonic Mxx-61-W, Japan) with a moderate speed for 1 minute. 500 ml of distilled water was added and was left for 1 hour. The blended onions were then distilled using a vertical steam distillation unit. The extract produced was then kept in the refrigerator until use (Benkeblia, N., 2004).

Preparation of different concentrations V/V (50%, 25% and 12.5%) of onion extract:

5 cc of onion extract mixed with 5 cc of distilled water in a tube gives a 75% concentration. 2.5 cc of medicinal herb mixed with 7.5 cc of distilled water gives a 25 % concentration. 1.25 cc of medicinal herb mixed with 8.75 cc of distilled water gives a 12.5 % concentration.

Inoculation of culture media with reference strain:

The pure culture of S. mutans and S. sanguinis was sub-cultured in nutrient broth as follow: After cleaning the ampoule tip with alcohol, the lyophilized ampoule tip was broken. Its content was then added to Triptycase Soy Broth (TSB) media and was incubated in an appropriate atmosphere (H2:CO2:N2 10:10:80) at 37[degrees]C for 24 hours. Bacteria cultivated were then inoculated onto the Blood agar media. The media was incubated for 24 hours. Bacteria that could grow under such circumstances were identified as reference strains. For antimicrobial susceptibility testing the turbidity must be adjusted equivalent to a 0.5 McFarland standard. A 0.5 McFarland standard is comparable to a bacterial suspension of [10.sup.6] CFU/ml.

Disk diffusion method:

After preparing the different concentrations of onion extract, each sterile paper disk was impregnated with 30 [micro]l of diluted onion extract. The disks were allowed to dry. Using a sterile forceps, the disks were placed on the inoculated blood agar medium. 1 paper disk on each plate was soaked in distilled water as a negative control. Then the plates were incubated in an appropriate atmosphere ([H.sub.2]:C[O.sub.2]:[N.sub.2] 10:10:80) at 37[degrees]C for 24 hours and the diameter of the inhibition zone was measured by a caliper. Each test was done in triplicate and the average was reported (Murray, P., 1995).

Broth micro dilution assay:

For broth micro dilution, susceptibility panel in 96-well micro titer plates (12 columns and 8 rows) were prepared by dispensing 200 [micro]l of TSB to the first column and 100 [micro]l of TSB into the rest wells by an 8- channel pipette. The rows were labeled from A to H. Then, the two-fold serial dilutions of onion extract was made by drawing up 100 [micro]l of each solution from the second column wells into the third column and then move on to the next column until the eleventh column to achieve the final concentrations. This procedure was done for the rows C, D and E. the same method was used for vancomycin (32ug/ml) as an index for antibacterial activity for the rows F, G and H. Finally 100 [micro]l of each bacterial suspension were inoculated into wells of micro titer plates to obtain the final volume of 5 x [10.sup.5] CFU/ml in each well of the plate. The first and the last wells in each row were negative and positive controls, respectively. The positive control was inoculated with bacterial suspension only, while the negative control well was left blank without inoculation. The 96-micro well plates were sealed using a perforated plate seal and incubated in an appropriate atmosphere ([H.sub.2]:C[O.sub.2]:[N.sub.2] 10:10:80) at 37[degrees]C for 48 h. The MICs were recorded as the lowest concentration where no viability is observed in the wells of 96-micro well plates after incubation period. Each test was done in triplicate.

Mean values and standard deviations of the diameter of inhibition zone in disk diffusion assay were calculated from the experimental data obtained. Differences between the mean values were considered significant when p < 0.05. The data was statistically analyzed by applying Fisher's exact test to compare the groups using SPSS software (version 17, Chicago, IL, USA).

Results:

This experimental study was conducted to assess the antibacterial activity of Allium cepa extract and vancomycin on Streptococcus mutans and Streptococcus sanguinis. The results of the disk diffusion method for three types of onion and vancomycin are illustrated in table 1. In this method no inhibition zones were measured for the 12.5% and 25% concentration of onion extract (p < 0.001). The diameters of inhibition zone of the 50% concentration of three types of onion (yellow, red and green) against Streptococcus mutans were (2.2 [+ or -] 0.2), (3.5 [+ or -] 0.3), and (1.2 [+ or -] 0.2) mm respectively. No inhibition zones were observed for the 12.5%, 25% and 50% concentrations of yellow and green onion against Streptococcus sanguinis. The diameter of inhibition zone of the 50% concentration of red onion was (1.5 [+ or -] 0.1) mm. Onion extracts had more antibacterial activity against Streptococcus mutans than Streptococcus sanguinis (p < 0.002). The diameters of inhibition zones for 32%, 16% and 8% concentrations of vancomycin against Streptococcus mutans were (10 [+ or -] 0.1), (8 [+ or -] 0.3), and (6 [+ or -] 0.2) mm respectively, and against Streptococcus sanguinis were (8 [+ or -] 0.2), (7 [+ or -] 0.2), and (5 [+ or -] 0.3) mm respectively. This showed that as the concentration of the vancomycin increased, the antibacterial activity also showed increase. The antibacterial activity of red onion was more than yellow and green onions (p<0.001). Green onion had the least antibacterial activity. The results of the broth microdilution method for three types of onion and vancomycin are illustrated in table 2. The MIC values of yellow, red, and green onion, and vancomycin against Streptococcus mutans were 12.5 %, 6.25 %, 12.5 % mm and 4ug/ml, respectively. The MIC values of yellow, red and green onion and vancomycin against Streptococcus sanguinis were 25 %, 12.5 %, 50 % mm and 4[micro]g/ml, respectively.

Discussion:

Allium cepa has been widely studied for its antimicrobial activity. However a limited data is available so far regarding its antibacterial activity against Streptococcus mutans, and Streptococcus sanguinis.

The antibacterial activity of red type of Allium cepa extract was found to be better as compared to yellow and green types. Onion extracts had more antibacterial activity against Streptococcus mutans than Streptococcus sanguinis. The antibacterial activity of the extracts has been increased by increasing the concentrations.

In a study conducted by Benkeblia et al., red onion exhibited a better antibacterial activity than yellow one against Staphylococcus aureus and Salmonella enteritidis. The zone of inhibition increased with increasing concentration of extracts (Benkeblia, N., 2004).

Kim et al. reported antibacterial activity of onion extract against Streptococcus mutans, Streptococcus sobrinus, Prevotella intermedia and Porphyromonas gingivalis which is similar to our results (Kim, J.H., 1997).

Maidment et al. examined a total of twelve Alliums for antibacterial activity against Escherichia coli. Allium cepa did not show antibacterial activity (Maidment, D., 2001). This different result compared to our study is explainable. Escherichia coli is a gram negative bacterium while Streptococcus mutans and Streptococcus sanguinis are both gram positives. Gram positives are proved to be more sensitive to essential oils and extracts than gram negatives (Shelef, L., 1984; Mann, C.M., 2000; Zohri, A.N., 1995; Sikkema, J., 1994).

It is evident from our study that red onion has a better antibacterial activity than yellow and green onions. This might be due to the differences in their compositions, differences in their growth climate. It is also proved that the constituents of onion vary as the age of the onion increases (Briggs, W.H., 2002). The differences in the temperature while preparing agar plates may also affect the results.

It is hoped that this study would lead to the establishment of new and potent antimicrobial agent. However, further studies are recommended considering the age and the region of the onion against oral pathogens.

Conclusion:

It is concluded that Allium cepa extract has a more significant antibacterial activity against Streptococcus mutans that Streptococcus sanguinis. Therefore, Onion extract can be considered as an antibacterial agent to prevent human dental caries.

Acknowledgment

Authors would like to appreciate the financial support of Shiraz University of Medical Sciences.

References

Bauer, A.W., W.M. Kirby, J.C. Sherris, M. Turck, 1966. Antibiotic susceptibility testing by a standardized single disk method. American Journal of Clinical Pathology, 45(4): 493-6.

Benkeblia, N., 2004. Antimicrobial activity of essential oil extracts of various onions (Allium cepa) and garlic (Allium sativum). LWT--Food Science and Technology, 37(2): 263-68.

Briggs, W.H., J.D. Folts, H.E. Osman, H. Xiao, K.L. Parkin and I.L. Goldman, 2002. Variation in economically, and ecologically important traits in onion plant organs during reproductive development. Plant, Cell and Environment, 25(8): 1031-37

Caufield, P.W., A.P. Dasanayake, Y. Li, Y. Pan, J. Hsu, J.M. Hardin, 2000. Natural history of Streptococcus sanguinis in the oral cavity of infants. Infect Immun., 68(7): 4018-23.

Cohen, M.L., 2000. Changing patterns of infectious disease. Nature, 406(6797): 762-7.

Gonzalez, C., M. Rubio, J. Romero-Vivas, M. Gonzalez, J.J. Picazo, 1999. Bacteremic pneumonia due to Staphylococcus aureus: A comparison of disease caused. Clinical Infectious Disease, 29(5): 1171-7.

Kim, J.H., 1997. Anti-bacterial action of onion (Allium cepa L.) extracts against oral pathogenic. Journal of Nihon University School of Dentistry, 39(3): 136-41.

Kuramitsu, H., 1993. Virulence Factors of Mutans Streptococci: Role of Molecular Genetics. Critical Reviews in Oral Biology & Medicine, 4(2): 159-76.

Lampe, J.W., 1997. Health effects of vegetables and fruit: assessing mechanisms of action in human. American Journal of Clinical Nutrition, 70(3 Suppl): 475s-90s.

Maidment, D., Z. Dembny, D. Watts, 2001. The antibacterial activity of 12 Alliums against E. coli. nutrition & food science, 31(5): 238-41.

Mann, C.M., S.D. Cox, J.L. Markham, 2000. The outer membrane of Pseudomonas aeruginosa NCTC 6749 contributes to its. Lett Appl Microbiol., 30(4): 294-7.

Murray, P., 1995. Manual of clinical microbiology. 6th ed. Washington, D.C: ASM press, pp: 1482.

Roberson, T., H.O. Heymann, 2006. Sturdevant's Art and Science of Operative Dentistry. 5th ed: mosby, pp: 69.

Shelef, L., 1984. Antimicrobial effect of spices. journal of food safety, 6(1): 29-44.

Sikkema, J., J.A. de Bont, B. Poolman, 1994. Interactions of cyclic hydrocarbons with biological membranes. Journal of Biological Chemistry, 269(11): 8022-8.

Small, P.M., H.F. Chambers, 1990. Vancomycin for Staphylococcus aureus endocarditis in intravenous drug users. Antimicrobial Agents Chemotherapy, 34(6): 1227-31.

Zohri, A.N., K. Abdel-Gawad, S. Saber, 1995. Antibacterial, antidermatophytic and antitoxigenic activities of onion (Allium cepa L.) oil. Microbiology Research, 150(2): 167-72.

(1) Ebrahimi H., (2) Bazargani A., (1) Pourshahidi S., (3) Rafiee A., (3) Gavahi M.

(1) Department of Oral Medicine, Dental School, Shiraz University of Medical Sciences, Shiraz, Iran

(2) Department of Bacteriology and Virology, Shiraz Medical School, Shiraz University of Medical Sciences, Shiraz, Iran

(3) Dental school, Shiraz University of Medical Sciences, Shiraz, Iran

Corresponding Author: Pourshahidi S., Department of Oral Medicine, Dental School, Shiraz University of Medical Sciences, Shiraz, Iran

E-mail: SaraPourshahidi20@hotmail.com
Table 1: Antimicrobial activity of various onion extracts and
vancomycin determinate by disk diffusion assay.

                                         Inhibition zone (mm)

variable     Concentration (%)    Streptococcus      Streptococcus
                                      mutans           sanguinis

Onion
                    50           2.2 [+ or -] 0.2          N*
Yellow              25                  N                  N
                   12.5                 N                  N

                    50           3.5 [+ or -] 0.3   1.5 [+ or -] 0.1
Red                 25                  N                  N
                   12.5                 N                  N
                    50           1.2 [+ or -] 0.2          N

green               25                  N                  N
                   12.5                 N                  N

vancomycin          32           10 [+ or -] 0.1     8 [+ or -] 0.2
                    16            8 [+ or -] 0.3     7 [+ or -] 0.2
                     8            6 [+ or -] o.2     5 [+ or -] 0.3

N = no inhibition zone

Table 2: MIC values of various onion extracts and vancomycin
determinate by broth microdilution assay.

Variable                         MIC

             Streptococcus mutans   Streptococcus sanguinis

Onion
Yellow              12.5%                     25%
Red                 6.25%                    12.5%
Green               12.5%                     50%
vancomycin      4 [micro]g/ml            4 [micro]g/ml
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Title Annotation:Original Article
Author:Ebrahimi H.; Bazargani A.; Pourshahidi S.; Rafiee A.; Gavahi M.
Publication:Advances in Natural and Applied Sciences
Date:Oct 1, 2012
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