The effect of aspartame and saccharin on the antimicrobial activity of chlorhexidine against mutans streptococci.

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1 The effect of aspartame and saccharin on the antimicrobial activity of chlorhexidine against mutans streptococci. Abbas S, Al Mizraqchi B.Sc, M.Sc, Ph.D. (1) Zainab A, Al Dhaher B.Sc, M.Sc. (2) Fadia Abd Muhsin B.Sc, M.Sc. (2) ABSTRACT Background: Although chlorhexidine is the most effective against dental plaque it is extremely bitter to prepare formulation, it is necessary to use flavoring and sweetening which can inhibit the antibacterial effect of rinse preparation. Materials and Method: The effect of different concentrations of aspartame and saccharin on the antibacterial effect of chlorhexidine.2% against Mutans Streptococci was evaluated using the agar diffusion method and determination of MIC value of chlorhexidine alone and in presence of aspartame or saccharin was also done in this study. Results: The results of this study demonstrate that at a concentration of %, 8%, 12%, 1% aspartame did not significantly inhibit the antibacterial activity of.2% chlorhexidine however the antibacterial activity of chlorhexidine significantly reduced when increased the concentrations of aspartame up to % and MIC values increased when increased the concentrations of aspartame, while saccharin interfere with the activity of chlorhexidine.2% and significantly reduced the anti mutans activity of chlorhexidine.2% at all the concentrations used in this study (% - %) and MIC values increased when increased the concentrations of saccharin. Conclusion: This in vitro study suggests that aspartame may be used as sweetener and flavoring in concentrations up to 1% with chlorhexidine rinse. Key words: Chlorhexidine, aspartame, saccharin, mutans streptococci. (J Bagh Coll Dentistry 28; 2(2):93-9) INTRODUCTION It is know that Mutans Streptococci (MS) are the principle a etiological agents of dental caries (1). Cariogenic features of these bacteria include synthesis of water insoluble glucans, lactic acid production, ability to survive at allow ph, intracellular polysaccharide synthesis and the production of dextrin hydrolyzing enzyme (endodextranase) (2). Mutans streptococci were found to be the predominant bacteria in caries process (3). Chlorhexidine (CHX), a bisbiguanide, has bactericidal activity against both gram- positive and gram-negative bacteria. Its effect against MS is greater than against other oral flora such as Streptococcus sanguis and lactobacilli (). Because chlorhexidine is positively charged it binds to various surfaces including enamel pellicle, hydroxyapatite and mucosa membranes. It binds to surface and disrupts bacterial cytroplasmic membranes, including leakage of low molecular weight components and the precipitation of cell content. (1) Assistant professor, Department of Basic sciences, College of Dentistry, University of Baghdad. (2) Assistant lecturer, Department of Basic sciences, College of Dentistry, University of Baghdad. Chlorhexdine inhibits key metabolic enzymes such glucosyltransferase, phosphoenolpyurate and phosphotransferase of MS (). A major part of the effectiveness of chlorhexidine is due to its substantivity, however, side effects of its use include discoloration of teeth and taste disturbances (). Aqueous and alcoholic solution of (CHX) can be prepared from a stock solution. Distilled water should be used because anions in tap water (chlorine, sulphate), can precipitate the bactericide. The problem of precipitation may also be encountered when coloring agents are added by the user to identify the concentration or to distinguish between aqueous and alcoholic solution. Soaps and other anionic detergents react with chlorhexidine and inactivate it (). Because CHX has an extremely bitter taste, it is often necessary to flavor and sweeten mouth rinses and gel product. Saccharin is considered compatible with CHX (8) and has been used in gel preparations (9,1). It has a structure similar to sulfonamide it is 3 times sweeter than sucrose, but it has a bitter taste when used at a concentration greater than.1 percent (11). Aspartame a dipeptide comprising aspartic acid and phenylalanine is 2 times sweeter than sucrose and is used extensively in sugar-free soft drink and chewing-gum (11,12). Basic Sciences 93

2 MATERIALS AND METHODS Stimulated salivary samples were collected under standard conditions (13). Ten volunteers with no medical history aged 21-3 years were selected to be participated in this study. Each individual was asked to chew a piece of Arabic chewing gum (.-. gm) for five minutes to stimulate salivary collection as much as possible then saliva was collected in sterilized screw capped bottles. After that, saliva was homogenized by vortex mixer for two minutes. Ten fold serial dilutions were prepared using sterile-normal saline (.1 ml) was withdrawn from dilution (1-2, 1 - ) and then spread in duplicate by using sterile microbiological spreader on plates of mitis salivarius agar (a selective media for mutans streptococci). Then the plates were incubated anaerobically for 8 hours at 3 C followed by aerobic incubation for 2 hours and 3 oc. The identification of MS based on the colony morphology under dissecting microscope, grams stain, fermentation of manitol (1). Cystine trypticase agar (CTA) had been used to test the ability of MS to ferment mannitol which was added in a concentration of 1% CTA. Pure broth cultures of M.S. were prepared using trypton soya broth (TSB) incubated aerobically for 2 hours at 3c. The activation of MS broth cultures were accomplished by transfer of.1 ml of the culture to sterile TSB and incubated aerobically at 3C for 2 hours. Final concentration of aspartame %, 8%, 12%, 1%, 2%, 2%, 28%, 32%, 3%, % and saccharin %, 12%, 18%, 2%, 3%, 3%, 2%, 8%, %, % where used to study the effect of aspartame and saccharin on the antimicrobial activity of chlorhexidine against MS using agar diffusion method technique. Seven isolates of MS were used in this study. The minimum inhibition concentration was determined. Final concentrations of chlorhexidine 1-1 µg/ml in trypton soya agar (TSA) in the presence of aspartame or saccharin were used to determine the MIC which is the lowest concentration of chlorhexidine that inhibit about 9% of the growth on TSA. Data were express as mean±standard deviation and a statistical significance was calculated by mean of ANOVA test (1). RESULTS The values of inhibitory zones of CHX with aspartame are shown in table 1. Table 1: Effect of chlorhexidine and chlorhexidine with aspartame on the growth of Mutans Streptococci. Mean ± Standard Deviation CHX Concentrations of aspartame.2% A1 A2 A3 A A A A A8 A9 A1 Mean of diameter control % 8% 12% 1% 2% 2% 28% 32% 3% % of Inhibition zone SD t - test p - value Sig NS NS NS S HS HS HS HS HS HS *p>. Non significant. **p<.1 Highly significant. ANOVA table F-test = P<.1 Highly significant. *A = aspartame. At concentrations of %, 8%, 12%, 1% Aspartame did not significantly inhibit the antibacterial activity of.2% CHX. However the antibacterial activity of CHX.2% was significantly reduced when the concentrations of aspartame were increased up to % (Figure 1). The means of the diameter of inhibition zone of CHX against MS were 3.3 mm, 3. mm, 3. mm, 32.1 mm, 29.9 mm, 2. mm, 23.3 mm, 21.1 mm, 2. mm, 19.9 mm, in presence of %, 8%, 12%, 1%, 2%, 2%, 28%, 32%, 3%, % aspartame respectively. These data were confirmed by determination of MIC of CHX alone and compared in the presence of CHX- Aspartame as shown in table 2 and the results showed that the MIC value increased when the concentration of aspartame was increased (Figure 3). Basic Sciences 9

3 concentration % Control A1 A2 A3 A A A A A8 A9 A1 diameter of Inhibition zone (mm) Figure 1: The effect of aspartame on the antibacterial activity of chlorhexidine in agar diffusion method. Table 2: Mean of MIC values of chlorohexidine (µg/ml) in presence of Aspartame. Bacterial Isolates frequency *CHX alone MIC of CHX (µg/ml) With: *A1 *A2 *A3 *A *A *A *A *A8 *A9 *A1 % 8% 12% 1% 2% 2% 28% 32% 3% % M.S M.S M.S M.S M.S M.S M.S Mean *CHX= chlorohexidine. *A= aspartame. On the other hand, saccharin interfered with the activity of CHX and significantly reduced the anti mutans activity of CHX. The mean of the diameter of inhibitions zones of CHX as shown in table 3 were 2. mm, 2. mm, 2. mm, 2. mm, 23. mm, 2. mm, 2 mm, 1.9 mm, 1 mm, 1. mm, in the presence of %, 12%, 18%, 2%, 3%, 3%, 2%, 8%, %, % saccharin respectively (Figure 2) and MIC values increased as shown in table when increased the concentration of saccharin; this is demonstrated in figure 3. Table 3: Effect of chlorhexidine and chlorhexidine with saccharin on the growth of Mutans Streptococci. Mean ± Standard Deviation CHX Concentrations of saccharin.2% S1 S2 S3 S S S S S8 S9 S1 Mean of diameter control % 12% 18% 2% 3% 3% 2% 8% % % of Inhibition zone SD t test p value Sig HS HS HS HS HS HS HS HS HS HS *p<.1 Highly significant. ANOVA table F-test = P<.1 Highly significant. S = saccharine. Basic Sciences 9

4 concentration of aspartame or ssacharin % Control S1 S2 S3 SA S S S S8 S9 S1 diameter of Inhibition zone (mm) Figure 2: The effect of saccharin on the antibacterial activity of chlorhexidine in agar diffusion method. Table : Mean of MIC values of chlorhexidine (µg/ml) in presence of saccharin. Bacterial isolates MS1 MS2 MS3 MS MS MS MS mean Frequency *CHX alone *S1 % MIC of CHX *S2 12% *S3 18% *S 2% *S 3% *S = saccharin. (µg/ml) *S 3% With: *S 2% *S8 8%..... *S9 %.8.2. *S1 % MIC ( µg/ml) CHX 3 Series1 Series concentration of aspartame or ssacharin % *CHX= chlorhexidine. Figure 3: MIC values of chlorhexidine alone and chlorhexedine with aspartame or saccharin against Mutans Streptococci. Basic Sciences 9

5 DISCUSSION Our results demonstrate that depending on concentration of %, 8%, 12% and 1% of aspartame we can use it to flavor and sweeten CHX.2%. It rinse without affecting anti mutans activity. These results agree with the results of Cury et al (1) who reported that when using CHX gel containing aspartame as a sweetener there was a reduction in salivary Mutans Streptococci. The results about saccharin demonstrated that at all the concentrations used in this study saccharin significantly reduced the antimutans-activity of CHX.2%. These results are in agreement with the results of the in vivo study of Rocha et al (1) that were not able to show reduced Mutans Setreptococci when 1% CHX gel containing 1% saccharin was used. The present in vitro evaluation about saccharin supports the observations of Van der Bijl et al (8) who prepared a mouth rinse containing.2% CHX and.1% saccharin in combination with.1% cyclamate to improve it taste. If saccharin had been the only sweetener added at a higher concentration, CHX may have been inhibited. The results also support the observations of Ostela et al (9) and tenovuo et al (1). In addition to all these Cury et al (1) reported that at a concentration of. and 1% saccharin significantly reduced the anti-mutans activity of 1% CHX gel they also reported that insoluble salts were formed between CHX and saccharin which may be through an interaction between the positively charged group of CHX and sulfonyl group of saccharin. These data were confirmed with our observations when precipitated insoluble products were observed when the CHX formulations were suspended in water it was possible to note that.2% CHX containing saccharin is cloudy while the CHX without saccharin is clear. We can also see these insoluble products in CHX.2% containing aspartame at concentrations 2%, 2%, 28%, 32%, 3%, % and CHX.2% with these concentrations of aspartame is also cloudy. This may explain why aspartame at these concentrations affects the anti-mutans activity of CHX.2%. In conclusion these invitro results suggest that saccharin inhibit the efficacy of chlorhexidine.2% while aspartame may be used as sweetener and flavoring CHX.2% rinse, in concentration up to 1%. 3. Samaranayake Lp, Jones BM, Swlly C. Essential Microbiology for Dentistry. 2 nd edition, Churchill Livingstone, 22.. Murray JJ, Nunn JM, Steet JG. The prevention of oral disease. th edition, Oxford University press, 23.. Sheie A. Chemoprophylaxis of dental curies, In Thylstrup A; Fejerskov.O. Eds. Textbook of clinical cariology.2 nd edition Copenhange munksgaard, 199: Blakrishnan M. Purification and characterization of mutacins produced by different clusters of inhibitory mutans Streptococci Ph.D. Thesis, Dunedin, university of Otago; Gardner JF, Peed MM, Kelsey JC. Churchill Livingstone, London Van der Bijl P, Dreyer WP. Chlorohexidine gluconate mouthrinse further aspect concerning its chemical compatibility, stability and detection of potentially harmful degradation products. J Dent Assoc South Africa 1982; 3: Ostela I, Tenovuo J, Soderling E, Lammi E, Lammi M. Effect of chlorohexidine sodium fluoride gel applied by tray or by toothbrush on salivary mutans streptococci. Proc Finn Dent Soc 199; 8: Tenovuo J, Hakkinen P, Paunio P, Emilson CG. Effect of clorhexidine fluoride gel treatments in mothers on the establishment of mutans streptococci in primary teeth and the development of dental caries in children. Caries Res 1992; 2: Rugg Gunn AJ. Nutrition and dental health. New York: Oxford university press, Bowen WH. Food components and caries. Adv Dent Res 199; 8: Dasanayake AP, Caufield PW, Cutter GR, Roseman JM, Köhlar B. Differences in the detection and enumeration of mutans streptococci due to differences in methods. Arch's Oral Biol 199; : Fingold S, Baron E. Method for identification of etiologic agents of infections disease. In. Bailey and Scott's diagnostic microbiology th edition C.V. Mosby co., St. louis Zar, JH. Biostatical analysis 2 nd ed. Prentice Hall Inc.Englewood, cliffs N.J. (198). 1. Cury JA, Rocha EP, Francisco SB, Del Bel cury AA. Effect of saccharin on antibacterial activity of chlorohexidine gel. Braz Dent J 2; 11:1, 29, Rocha EP, Francisco SB, Del Bel cury AA, Cury JA. Oral rehabilitation with removable prosthodontics and chemical control of mutans streptococci SBPqO. (Brazillian Division of IADR) 1999; 1: REFERENCES 1. Hamada S, Slade HD. Biology, immunology and cariogenicity of streptococcus mutans. Microbial Rev 198; : Van Hout J. Role of microorganisms in caries etiology. J Dent Res 199; 3: Basic Sciences 9