Antibacterial Activity of Alcoholic Cinnamon Extract and Chlorhexidine on Streptococcus Mitis as Primary Colonizers in Periodontal Disease

Ahmed Abdulwahid Hasan and Ayser Najah^*

^*Correspondence: Ayser Najah, Department of Periodontics, College of Dentistry, University of Baghdad, Iraq, Email:

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Introduction

Periodontal diseases are one of the most common diseases affecting human beings, the diseases start with bacterial adherence and the development of biofilms on the surfaces of the tooth including either natural or restored [1]. Dental plaque is pale yellowish in color biofilm which forms naturally on the teeth. A dental plaque is formed by colonizing bacteria attempting to attach themselves to the surface of the tooth. Dental biofilm, commonly referred to it as dental plaque, is a mixture of about a thousand bacterial species that constitute part of the sophisticated ecosystems of the mouth [2]. Dental plaque begins when present bacteria attach to teeth and start multiplying. Plaque may build on teeth either in supragingival plaque, or subgingival plaque, or both [3]. The plaque is built of various colonies of microorganisms such as bacteria, yeast stuck together in a gel-like organic material matrix composed of bacterial byproducts comprising sugar, food debris, and body tissue [4]. The dominant initial colonizers of teeth are Gram-positive facultative anaerobic cocci and rods, involving Streptococcus and Actinomyces species. These initial colonizers make a foundation for further development of dental biofilm [5]. Streptococcus mitis, Streptococcus oralis, and Streptococcus sanguinis of the Mitis group streptococci [6,7] are closely correlated to Streptococcus pneumonia, the important human pathogen.

The streptococcus species are part of human normal oral commensal flora, but sometimes, they cause acute or chronic episodes of disease as opportunistic human pathogens, unlike S. pneumonia. They are linked with gingival disease and caries and now and then cause sub-acute infective endocarditis [8]. Because they are considered as the early colonizers in the development of the dental biofilm [9]. They can gain natural competence for hereditary transformation as S. pneumonia [10] and have horizontal transferring of the gene, such as virulent genes, that has been recorded between these species [6,11-13]. The emergence of multidrug resistant (MDR) microorganisms has turned to be a danger to general health [14-16]. This worrisome lead to an immediate urge for the invention of contemporary antibacterial and treatment strategies.

Cinnamon's unique healing abilities come from three basic types of components found in its bark. These oils contain active components called cinnamaldehyde, cinnamyl acetate, and cinnamyl alcohol, plus a wide range of other volatile substances [17]. Cinnamon has been used in food preservatives owing to its antioxidant properties, antibacterial, insecticidal, and nematicidal activities. It has been widely used in the food industry. Also, it is employed in seasonings, sauces, bakery, confectionery, and drinks [18].

Cinnamaldehyde inhibits cell wall biosynthesis and causes cell membrane dysfunction [19]. The presumed mechanism of antimicrobial action of Cinnamon is probably due to the presence of Cinnamaldehyde, an aromatic aldehyde, which has electronegative and interferes in biological processes including electron transfer and reacts with nitrogen having components, for example, proteins and nucleic acids, and therefore stops the growth of the microorganisms, Most of the studies on cinnamon suggest that it is not harmful to humans and may be used as an agent to inhibit the growth of bacteria, fungi, and yeast [20].

Aim/Objectives

To evaluate the antibacterial effect of alcoholic cinnamon extracts on Streptococcus mitis bacteria.

To study the combination between Chlorhexidine and alcoholic cinnamon MIC when mixed.

Materials and Methods

Study design/sample

The material and methods were made through two steps:

The first step was the preparation of the cinnamon extract,

The second step was the preparation of the bacteria (streptococcus mitis)

First, preparation of the cinnamon

The type of cinnamon used in this study was SirLanka type which was bought from a local attar market (Jannat Alashab). The extraction was prepared at the University of Baghdad/ College of Agriculture/Department of medical herbs/University of Baghdad. The extraction process was made using the maceration process. Figures 1 and 2.

Figure 1: Showed cinnamon bark.

Figure 2: Cinnamon powder.

Second, The preparation of bacteria

Bacterial samples were taken from supragingival plaque through cotton swabs. A group of 20 volunteers was volunteered to give a sample then bacterial isolation, culturing, identification, and activation were done.

Procedure

The procedure was done according to the method of Shtayeh and Abu ghadeib.

The bark was ground using a grinding machine (Silvercrest mixer) into a fine powder. Then the powder was weighed using an electronic balance (SF-400) 20 gram of powder was placed in a glass beaker of 500 ml size and then 200 ml 70% ethyl alcohol was added to it, it was then placed in a shaker incubator (Binder BF-720) at 35 °C for 24 hours and it was filtered using Whatman filter paper No.1, then the product was centrifuged at 3000 rpm for 10 minutes, then it was dried in the oven (Oven at 40 °C.), then kept in dark and tight tubes in the freezer at a temperature of -18 °C until use.

Determination of the minimum inhibitory concentration (MIC)

The minimum inhibitory concentration (MIC) was defined as the lowest concentration of any anti-microbial agent that would inhibit the visible growth of the selected micro-organism [21-23]. The determination of the MIC was done by using the broth dilution method, it is one of the most basic antimicrobial susceptibility methods. This procedure involves preparatory two folds dilution of the cinnamon extract used (16 %, 8 %, 4 %, 2%, and 1%) in Muller Hinton broth dispersed in tubes containing a minimum volume of 2 ml, then, each tube is inoculated with microbial inoculum (S. mitis) prepared in the same medium of standardized microbial suspension adjusted to 0.5 McFarland scale. After well mixing, the inoculated tubes are incubated aerobically at 37 C for 24 hours. After 24 hours the tubes were checked by the naked eye. The tube that showed turbidity meant that there was bacterial growth and were excluded, while the tubes that revealed a clear vision were meant as no bacterial growth. The first tube that lacks turbidity was the MIC concentration.

Preparation of bacteria

A cotton swab was taken from supragingival plaque. A group of 20 volunteers with moderate oral hygiene were selected. The samples were put into a plane tube continuing 10 ML of brain heart infusion broth. Isolation of the streptococci is performed through homogenizing plaque samples by a vortex mixer for two minutes, then ten-fold dilution was performed by transferring 0.5 ml of saliva to 4.5 ml of phosphate buffer saline (pH 7.0). From dilution 10-3, 10-4, and 10-5 of collected samples, 0.1 was taken and spread in duplicate on the MSB agar media, the plates were incubated anaerobically using a candle for 48hr. at 37º C then aerobically for 24 hr. at room temperature. The identification of streptococcus mitis was done by morphological and biochemical characteristics, in a sterile condition, the colony was taken from the blood agar plate and exposed to Grams stain.

Grams stains

The single isolated colony mixed with a drop of distal water on a glass slide, then spread, dried, then fixed with heat, after that coated with crystal violet for 1 min, then rinsed with distal water, coated with Lugols iodine for 1 minute then rinsed, decolorized with acetone for 30 seconds and rinsed, finally stained with safranin for 1 minute then rinsed and dried (Figure 3).

Figure 3: Gram-stained bacteria under the microscope.

A blood Hemolysis test used to evaluate the capability of Streptococcus mitis to bleach the heme iron via hydrogen peroxide (H2O2), forming to greenish color on the blood agar [24].

Catalase production test

This test was achieved on colonies of bacteria. Hydrogen peroxide 3% (H2O2) had been used to evaluate the ability to create catalase enzyme by bacteria [25].

Vitek 2 machine

Vitek 2 for identifying the specific bacterial type (Table 1 and Figure 4) [26].

Figure 4: Vitek 2 machine.

Table 1: Streptococcus mitis identification by Vitek 2.

The experiment of the study

Antimicrobial effect of Streptococci mitis, St. sanguinis, and St. Salivarius to different Concentrations of Cinnamon Extracts, Chlorhexidine, and Deionized Water in Vitro.

Different concentrations of cinnamon extract and were used in this experiment, the concentrations were as follows: 1.5%, 2.5%, 5%, and 10%. The Agar well technique was used in this experiment to study the sensitivities of bacteria to these agents on Mueller Hinton agar on ten isolates of streptococcus species (Figure 5).

Figure 5: Testing antimicrobial effect of the cinnamon extract on St. mitis.

Procedure

â??A volume of 25 ml of MHA was poured into sterile glass Petri dishes, left at room temperature for 24 hr.

â??To each plate 0.1 ml of streptococci species inoculum was spread, left for 20 minutes at room temperature then wells of equal size and depth were prepared about (three-four wells) in each plate, each well was filled with 0.2ml of the test agents.

â??Plates were left at room temperature for one hour then incubated anaerobically for 24 hr. at 37°C, each zone of inhibition was measured across the diameter of each well. No zone of inhibition indicated complete resistance of bacteria to the test agent.

Test agents

â??99 Cinnamon extracts :( 1.25%, 2.5%, 5% and 10%). â??99 CHX gluconate 0.2%. â??99 DW.

Results

Agar well diffusion method was used to determine the sensitivity of S. mitis to different concentrations of alcoholic cinnamon extracts, CHX (0.2%), and D.W. in vitro. The diameter of the inhibition zone (clear zone which represents no growth of S. sanguinis around each well) was found to increase as we increased the concentration of cinnamon extracts (Figures 6 and 7).

Figure 6: Sensitivities of S. mitis to different concentrations of alcoholic cinnamon extract.

Figure 7: Sensitivities of S. mitis to the combination of the alcoholic cinnamon extract MIC with CHX and CHX alone.

Determination of the minimum inhibitory concentration (MIC)

Determination of the minimum inhibitory concentration (MIC) was done using the Broth dilution method (Figure 8).

Figure 8: MIC of alcoholic cinnamon extract.

The MIC value for S. mitis was

The alcoholic was 2% (0.2 mg/ml)

The chlorhexidine (CHX) showed no turbidity which meant the absence of bacterial cell growth (S. mitis) at 0.2% concentration.

D.W. showed no turbidity which meant the absence of bacterial growth.

Figure 9 showed the results of alcoholic cinnamon extracts. Alcoholic cinnamon extracts were used by four different concentration (1.25%, 2.5%, 5% and 10 %), CHX (0.2%) as positive and distilled water (D.W). as negative control. The inhibition zone for an alcoholic was measured. The 1.25% cinnamon extract gave, as well as the distilled water, 0 mm. The concentration of 2.5 % gave a 15 mm inhibition zone (clear circle around the well in the agar disk). The concentration of 5% gave us a 17 mm inhibition zone. The concentration of 10% gave us 20 mm. the CHX by itself only gave us 18 mm. The combination of alcoholic cinnamon MIC (2%) and CHX gave us a 22 mm inhibition zone. the results of the different concentrations used in the sensitivity test revealed that the mixture of the alcoholic cinnamon extract and CHX gave the highest inhibition number 22 mm, followed by CHX 20 mm. The mixture of alcoholic cinnamon MIC and CHX showed that the cinnamon had improved the activity of CHX.

Figure 9: Growth inhibition zone against S. mitis bacteria in response to the concentrations of alcoholic cinnamon, CHX, and distilled water.

Table 2 showed the comparison in the mean of growth inhibition zone between different concentrations of cinnamon, CHX, and the combination of cinnamon MIC and CHX using the ANOVA test. This was specifically done against the S. mitis. The result was significantly higher (P<0.05). It was clear that there was a difference in the effect among the different concentrations that were used against S. mitis, the different preparations did not give the same inhibition number, as we mentioned above, the inhibition zone increased as we increased the concentration.

Table 2: Comparison in the mean of growth inhibition zone against S. mitis bacteria between different concentrations and preparations of cinnamon and CHX.

Post hoc tests or least significant value (LSD) was run to confirm the differences occurred in growth inhibition zone between different preparations and concentrations of cinnamon and CHX 2% and showed that in alcoholic preparation, mean of growth inhibition zone that developed by mixture of cinnamon and 2% CHX was significantly higher (P<0.05) than that in all other concentrations except 10% cinnamon when it was not significantly different (P=0.095). Also, it was increased by increasing the concentration of cinnamon except in concentrations of 2.5% and 5% when it was not significantly different (Table 3).

Table 3: Post hoc tests to confirm the differences occurred in the growth inhibition zone of S. mitis bacteria between different preparations and concentrations of cinnamon and CHX 2%.

Discussion

Cinnamon has been used by humankind for thousands of years [27-29], for cooking, medical purposes for perfumes [30], the unique, sweet, and distinguishable odor made cinnamon one of the luxurious spices that have been used by rich people [31,32].

Contemporary medicine focuses on natural materials that safe for humans and animals, friendly to the environment, and effective against pathogens. Plants and herbs are a natural material that grows on earth without chemical reactions intervention [33].

Cinnamon extracts cause inhibition of bacterial growth in many ways. The action of cinnamon could be due to the predominant active component present in cinnamon which is cinnamaldehyde and cinnamyl acetate which leads to inhibition of proton motive force, the chain of respiration, transfer of the electron, and substrate oxidation, resulting in unbinding of oxidative phosphorylation, active transport inhibition, loss of pool metabolites, and damages to the synthesis of DNA, RNA, proteins, polysaccharides, and lipids [34]. In addition to that, the property of the extract of cinnamon and its components is its hydrophobicity, which enables it to disturb the lipid bilayer of the membrane of the cell, rendering the wall more permeable to protons. Extensive leakage from bacterial cells or the exit of critical molecules and ions ultimately leads to bacterial cell death [35].

Chlorhexidine is the most known agent that has been used as a chemical plaque controlling agent. It has been reported to be the most active agent used in mouthwash. As well as CHX is one of the most important known antiseptics, low concentration is bacteriostatic and on high concentration, it is bactericidal [36]. In addition to that, CHX affects a broad range of bacteria covering gram-positive and gram-negative and even including some fungi [37]. The previous findings have shown that washing with 10 ml of CHX gluconate solution 0.2%, twice a day, almost completely stops the progress of the microbial plaque in humans. Therefore, gingivitis and periodontitis can be prevented, and this is confirmed by empirical studies. Clinical studies that used CHX for several months as a mouthwash showed a 45 to 61% decrease of the microbial plaque and a 27 to 67% decrease in gingivitis [38]. CHX does not interact with any microbial enzymes or receptors and hence does not lead to resistance from organisms [39].

Oral Streptococci of the Mitis group Streptococci [40,41], as S. mitis, S. sanguinis, and S. salivarius, which are mutually related to the S. pneumonia (the significant human pathogen). They are initial colonizers in the development of the biofilms of the oral cavity [42], Streptococcus mitis, Streptococcus sanguinis, and S. salivarius were chosen in the presented study due to their function in the development of plaque and eventually the periodontal disease [43]. In addition to the fact that they are the most found bacteria of dental plaque as primary plaque colonizing bacteria [44].

The explanation for the synergism between the extract of cinnamon and CHX could be because the CHX is a dicationic molecule with has high tendency to attach to an anionic molecule, the cell membrane is anionic where CHX bind to it leading to disruption of the cell wall, the disruption of the bacterial cell wall enhances the entry of cinnamon molecule inside the cell in higher quantity leading to more effective action [45-47]. Once the cinnamon gets inside the bacterial cell, it will cause proton motive force, the chain of respiration, transfer of the electron, and substrate oxidation resulting in unbinding of oxidative phosphorylation, active transport inhibition, loss of pool metabolites, and damages to the synthesis of DNA, RNA, proteins, polysaccharides, and lipids [34].

Conclusion

Cinnamon has a potent antibacterial effect against Streptococcus mitis. The combination between CHX and cinnamon is synergistic. Curcumin can be a promising therapeutic agent as an alternative to CHX.

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